Human-derived anti-huntingtin (htt) antibodies and uses thereof

ABSTRACT

Provided are novel human-derived anti-huntingtin (HTT) antibodies and biotechnological derivatives thereof, preferably capable of binding mutated and/or aggregated HTT species and or fragments thereof, as well as methods related thereto. The human-derived anti-HTT antibodies and biotechnological derivatives can be used in pharmaceutical and diagnostic compositions for HTT targeted immunotherapy of Huntington Disease and diagnosis thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 29, 2022, isnamed 51467-006003_Sequence_Listing_6_29_22_ST25 and is 378,721 bytes insize.

FIELD OF THE INVENTION

The present invention generally relates to antibody-based therapy ofHuntington's disease (HD) associated with Huntingtin (HTT). Inparticular, the present invention relates to novel moleculesspecifically binding to human HTT and/or antigens thereof, particularlyhuman-derived antibodies as well as HTT-binding fragments, synthetic andbiotechnological derivatives thereof, which are useful in the treatmentof diseases and conditions induced by such pathogenic HTT isoforms.

In addition, the present invention relates to pharmaceutical anddiagnostic compositions comprising such HTT-binding molecules,antibodies and mimics thereof valuable both as a diagnostic tool toidentify diseases and/or disorders associated with HTT aggregation andas a passive vaccination strategy for treating disorders related todiseases associated with HTT amyloidosis.

BACKGROUND OF THE INVENTION

Huntington's disease (HD) is an autosomal dominant neurologicalamyloidogenic disease. 5 to 10 individuals per 100,000 individuals areaffected with this autosomal disease. However, the prevalence in the USis much higher, studies have shown that under 200,000 US individuals 50%have the risk of developing HD, in particular 30,000 patients areregistered in the US while only 100,000 patients are registeredworldwide.

HD, as shown in several studies, results from a trinucleotide CAG repeatexpansion in the Huntingtin (HTT) gene, in particular in exon 1 of theHTT gene located on chromosome 4 (MacDonald et al., Cell 72, (1993),971-983), which is translated into a polyglutamine (polyQ) stretch inthe HTT protein. HD occurs when the polyQ tract exceeds a threshold of35-40 glutamine residues in length with a strong inverse correlationbetween repeat length and age-of-onset of disease. This polyQ stretchleads to a misfolding and aggregation of HTT in several regions, e.g.neurons and glial cells. With increasing age an accumulation of the HTTaggregates takes place leading to degeneration of the striatalGABA-ergic neurons and cortical pyramidal neurons. Symptoms of the HTTmisfolding and aggregation include involuntary movements, lack of motorcoordination, depression, cognitive decline such as memory loss and/ordementia.

Since 1993 when the HD mutation was identified the understanding of thepathophysiology and molecular biology of the disease has significantlyimproved. Medicaments such as e.g. Xenazine® (tetrabenazine, Lundbeck) ahexahydro-dimethoxy-benzoquinolizine derivative VMAT2 inhibitor had beendesigned for symptomatic treatment targeting involuntary musclemovements.

In addition, gene silencing approaches such as RNA interference (RNAi)have been suggested as potential therapies. In particular, the use ofsiRNA directed against HTT gene in a HD mouse model (R6/2) was shown toinhibit mutant HTT gene expression, see e.g. Warby et al., Am. J. HumGenet. 84 (2009), 351-366 and Olshina et al., Biological Chemistry 285(2010), 21807-21816. However, one limitation of this method lies in thedifficulty to introduce sufficient amount of siRNA into the target cellsor tissues as shown by e.g. Boudreau et al. (Brain Research 1338 (2010),112-121). Furthermore this approach may face safety liabilities as acontinued need for the expression of Huntingtin was suggested by genedeletions studies in animal models and cultured cells (Dragatsis et al.,Nat. Genet. 26 (2000), 300-306; Gauthier et al., Cell. 118 (2004),127-138; Zuccato et al., Nat. Genet. 35 (2003), 76-83).

Therefore, there is a need for novel therapeutic strategies anefficacious and safe therapy of diseases associated with HTT aggregationwhich preferably directly interfere with amyloid formation by mutantHTT.

This technical problem is solved by the embodiments characterized in theclaims and described further below and illustrated in the Examples andFigures.

SUMMARY OF THE INVENTION

The present invention provides anti-huntingtin (HTT) antibodies andequivalent HTT-binding molecules for use in the prophylactic ortherapeutic treatment of diseases and conditions associated with HTTamyloidosis. More specifically, therapeutically useful human-derivedantibodies as well as HTT-binding fragments, synthetic andbiotechnological derivatives thereof that recognize mutated and/oraggregated forms of HTT are provided.

In particular, experiments performed in accordance with the presentinvention were successful in the recombinant cloning and production ofhuman-derived monoclonal HTT-specific antibodies which are specific formutated and/or aggregated HTT species and/or fragments thereof. Thehuman subjects being the source of the B cells from which the cDNAencoding the variable domain of human-derived monoclonal anti-HTTantibodies, respectively, have been isolated, were healthy donors.However, in another embodiment of the present invention, the source ofthe B cells from which the human-derived monoclonal anti-HTT antibodiesand the cDNA encoding their variable domain, respectively, might beisolated are HD patients carrying trinucleotide CAG repeat expansion inthe HTT gene and being either symptom-free or displaying an unusuallyslow progressing or stable disease course or alternatively displayingtypical clinical features of Huntington's disease. Furthermore, asdemonstrated in the Examples, the antibodies of the present inventionare capable of attenuating dendritic spine loss, improve behavioralperformance during task-specific training and enhance sensorimotorability in a mouse model of HD. Therefore, it is prudent to expect thatthe human monoclonal anti-HTT antibodies of the present invention andderivatives thereof besides being non-immunogenic also exhibit atherapeutically beneficial effect in human.

As described in the background section, hitherto the pathogenesis of HDhas been tried block by intracellular approaches such as RNAinterference (RNAi); see also, e.g., Stanek et al., Human Gene Therapy25 (2014), 461-474 for silencing mutant Huntingtin by Adeno-associatedvirus-mediated RNA interference. With respect to an immunotherapeuticapproach the intracellular expression of single-chain antibody fragments(scFv), i.e. intrabodies which are devoid of the constant region ofimmunoglobulins such as of the IgG class has been explored in the lastdecade; see, e.g., supra and Butler et al., Prog Neurobiol. 97 (2012),for engineered intracellular scFv and single-domain (dAb; nanobody)antibody therapies to counteract mutant huntingtin and related toxicintracellular proteins.

For example, Lecerf et al., Proc. Nat. Acad. Sci. 98 (2001), 4764-4769describe a single-chain variable region fragment (scFv) antibodyspecific for the 17 N terminal residues of huntingtin, adjacent to thepolyglutamine in HD exon 1 selected from a large human phage display. Acorresponding scFv antibody, scFv-C4 comprising a lambda variable light(V_(L)) chain (Kvam et al., PLoS One 4 (2009), e5727; GenBank accessionnumber ACA53373) is described to have some neuro-protective effect inB6.CgHDR6/1 transgenic mice, a HD mouse model, which however weakenedboth with severity of disease at time of injection. In order to improvethe steady-level of the intrabody and to direct N-terminal htt exon 1(httexl) protein fragments bound by scFv-C4 to the proteasome fordegradation in order to prevent them from aggregation the PEST signalsequence of Mouse Ornithine Decarboxylase (mODC) mODC has been fused tothe scFv-C4 antibody; see Butler and Messer, PLoS One 6 (2011), e29199.No in vivo experiments have been reported yet.

Also the group of Khoshnan et al. was aiming at the development ofintrabody-based therapeutics for HD and inter alia describeanti-huntingtin scFv antibodies derived from mouse monoclonal antibodiesbinding the epitopes polyglutamine (polyQ), polyproline (polyP), andanti-C terminus and their effects upon intracellular expression onmutant huntingtin aggregation and toxicity; see, e.g., Ko et al., BrainResearch 56 (2001), 319-329, Khoshnan et al., Proc. Nat. Acad. Sci 99(2002), 1002-1007 and Legleiter et al., J. Biol. Chem. 284 (2009),21647-21658 and their patent application US 2003/0232052 A1. In the USapplication, also a “human” scFv antibody denoted “hMW9” is described tohave been isolated from a human scFvs phage library using recombinantmutant huntingtin protein. However, in contrast to mouse monoclonalderived scFv MW1, MW2, MW7 and MW8 no sequence data are provided forhMW9 which hitherto has also never been reported again.

Colby et al., Proc. Nat. Acad. Sci. 342 (2004), 901-912 describe thedevelopment of a human light chain variable domain (V_(L)) intracellularantibody specific for the amino terminus of Huntingtin via yeast surfacedisplay of a non-immune human antibody library. This single-domainintrabody consisting only of the lambda light chain domain of theoriginal scFv was described to inhibit huntingtin aggregation in acell-free in vitro assay as well as in a mammalian cell culture model ofHD; see also to corresponding international application WO 2005/052002.Again, no in vivo experiments have been reported yet.

Hence, apparently current intrabody based approaches either did notextend over cell-based assays or had not been proven to be successful inanimal models of HD yet, at least not in long term experiments. Inparticular, intrabodies reveal several limitations in-vivo such as theirpotential toxicity due to intracellular/intranuclear accumulation ofintrabody-antigen complexes or the limited distribution of for exampleviral delivery into large brain volumes in humans; see, e.g., Butler etal., Prog. Neurobiol. 97 (2012), 190-204 and Sothwell et al., J.Neurosci. 29 (2009), 13589-13602. Furthermore, a general drawback ofintracellular approaches is the problem of addressing the antibody andits encoding vector DNA, respectively, to the desired cells and, ifexogenously applied the inconvenient administration regimen, for exampleintrastriatal injections; see, e.g., Snyder-Keller et al., Neuropathol.Exp. Neurol. 69 (2010), 1078-1085. In addition, general concerns withrespect to gene therapy and the use of viral vectors remain.

In contrast, the experiments performed in accordance with the presentinvention demonstrate for the first time that full-length IgG antibodiesdirected against different epitopes of huntingtin upon systemicadministration can be successfully delivered to the brain (Example 24and FIG. 18) and that the antibodies of the present invention arecapable of attenuating dendritic spine loss, improve behavioralperformance during task-specific training and enhance sensorimotorability in a mouse model of HD (Example 34 and FIG. 34).

Therefore, as illustrated in the Examples, the anti-HTT antibody or anHTT-binding fragment, synthetic or biotechnological derivative thereofis preferably of the IgG class, which as generally known and describedherein comprises two identical variable heavy (V_(H)) chain polypeptidesand two identical variable light (V_(L)) chain polypeptides, and aconstant region and domain, respectively, i.e. at least one or all ofthe constant domains of the light chain (CL) and the heavy chain (CH1,CH2 or CH3). Put in other words, in one aspect of the present inventionrecombinantly expressed bivalent antibodies specific for Huntingtin andaggregated forms, fragments, peptides and derivatives thereof areprovided suitable for use in the treatment or in in vivo diagnosis ofhuntingtin and disorders associated therewith, which are characterizedby the presence of an immunoglobulin constant region. As describedherein further below the immunoglobulin may be of any class such as IgG,IgM, IgA IgG, or IgE and corresponding immunoglobulin subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1. Preferably however, theantibody is of the human IgG subtype.

In addition, as also further explained herein, the human-derivedantibodies of the present invention are characterized by comprising atleast one or more CDR of human origin, i.e. being encoded by a cDNAderived from human memory B cells, and preferably wherein the V_(H)and/or V_(L) chain are of human memory B cell origin too. The constantregion or any domain thereof if human may be of the same or differentorigin as the CDR(s) and the V_(H) and/or V_(L) chain, respectively.

In this context, unless stated otherwise or clear from context referenceherein to the antibody of the present invention includes thehuman-derived antibodies illustrated in the Examples as well asHTT-binding fragments, synthetic and biotechnological derivativesthereof.

As can be further noted form the prior art approaches of providingintrabodies derived from human scFvs phage library almost always scFcvswere obtained with a variable light chain of Vlamda origin; see Kvam etal. and Colby et al., supra. In contrast, more than 90% of thehuman-derived antibodies of the present invention use a Vkappa lightchain, which also applies to antibodies NI-302.31F11 and NI-302.35C1illustrated in Example 24 to be capable of penetrating the brain uponsystemic administration and in Example 34 (NI-302.35C1) to havebeneficial effects on behavioral performance and motor-related tasks ofmice in a HD animal model. Therefore, it is tempting to speculate thatantibodies having a Vkappa light chain might have superior propertiesover antibodies having a light chain of Vlamda origin. Therefore, in apreferred embodiment of the antibody of the present invention thevariable light chain is of Vkappa origin.

As illustrated in the Examples and Figures, the anti-HTT-antibody,HTT-binding fragment, synthetic and biotechnological variant thereofbinds to different regions of the HTT exon 1 protein which shows the“toxic” alteration as described above, i.e. the expanded, unstabletrinucleotide repeat, as shown in the Examples. In particular, theantibody of the present invention recognizes a polyP-region, apolyQ/polyP-region, the P-rich-region, the C terminal-region or theN-terminal region of HTT exon 1 protein. The epitopes of the subjectantibodies illustrated in the Examples are summarized in FIG. 20. Asmentioned in the background section, HD occurs when the polyQ tractexceeds a threshold of 35-40 glutamine residues in length due to anaggregation of HTT. Accordingly, as shown in Example 3, aggregated andsoluble HTT exon 1 proteins with 21, 35 or 49 polyQ repeats weregenerated and the binding of the identified antibodies tested. In thefollowing these constructs will be denoted HDX with X being the numberof Qs, e.g. HTT exon 1 with 21 polyQ repeats will be denoted HD21.Therefore, unless specifically indicated otherwise the term HTT meansHTT exon1 and the soluble HTT refers to the corresponding GST-fusionproteins.

In a preferred embodiment of the present invention, the anti-HTTantibody or HTT-binding fragment, synthetic or biotechnologicalderivative thereof is capable of preferentially binding aggregated ormisfolded forms of HTT. As described in e.g. Legleiter et al., JBC 285(19) (2010), 14777-14790 and demonstrated in the Examples theaggregation of HDX proteins in terms of speed and seize increases withthe number of Qs.

In a particularly preferred embodiment of the present invention, theanti-HTT antibody or HTT-binding fragment, synthetic or biotechnologicalderivative thereof demonstrates the immunological bindingcharacteristics of an antibody characterized by any one of the variableregions V_(H) and/or V_(L) as set forth in FIG. 1. Preferably, thevariable region of the antibody comprises at least one complementaritydetermining region (CDR) of the V_(H) and/or V_(L) of the variableregions, i.e. pair of V_(H) and V_(L) chain as set forth in FIG. 1A to1AU, wherein one or more amino acid substitutions are permitted as longas binding specificity of the resultant antibody compared to the subjectantibody comprising the corresponding pair of V_(H) and V_(L) chain asset forth in FIG. 1A to 1AU as illustrated in the Examples, e.g. assummarized in FIG. 20 remains unaffected in kind, i.e. epitopespecificity and EC₅₀ values in the same order of magnitude for theindicated antigen, preferably in the range of at least 50%, morepreferably 25% and most preferably at least 10% identical value.Preferably, one, two or all three CDRs of the V_(H) and V_(L) chaincontain at least one amino acid at a corresponding position which isconserved (i.e. being the same or a conservative substitute amino acid)in at least about 20%, preferably about 40%, more preferably about 50%and most preferably about 75% in the V_(H) and V_(L) chain amino acidsequences, respectively, of the subject antibodies which recognize thesame type of HTT epitope, i.e. poly-P, P-rich, C-terminus or N-terminus.For example, sequence alignment of the subject antibodies reveals thepredominant presence of one or two tyrosines (Y) in CDRH1; see FIG. 36.Similar conserved amino acid can be identified in the other CDRs aswell.

In a further embodiment of the present invention, the anti-HTT antibodyor HTT-binding fragment, synthetic or biotechnological derivativethereof is a bispecific antibody. Thus, the antibody of the presentinvention may be capable of recognizing at least two distinct epitopeseither on the same or on different antigens. For example, while a firstantigen-binding site, i.e. variable domain may be specific for HTT andpreferably comprises a variable region of any one of the subjectantibodies illustrated in the appended Examples and Figures, the secondantigen-binding site may be specific for a different, preferably alsoneurotoxic protein and comprise a variable region of correspondingantibody. Hence, protein misfolding and aggregation is a major hallmarkof neurodegenerative disorders such as Alzheimer's disease (AD),Parkinson's disease (PD) and HD. Tough until recently, the consensus wasthat each aggregation-prone protein was characteristic of each disorder[α-synuclein (α-syn)/PD, mutant huntingtin (HTT)/HD, Tau and amyloidbeta peptide/AD], growing evidence indicates that aggregation-proneproteins can actually co-aggregate and modify each other's behavior andtoxicity, suggesting that this process may also contribute to theoverlap in clinical symptoms across different diseases; see, e.g., forco-aggregation of α-syn and mutant HTT Poças et al., Hum. Mol. Genet. 24(2015), 1898-1907.

Thus, in one embodiment of the present invention the anti-HTT antibodyor HTT-binding fragment, synthetic or biotechnological derivativethereof is a bispecific antibody which is capable of binding HTT and aprotein associated with a neurodegenerative disorder, in particular inthe brain, preferably selected from the group consisting of α-synuclein,Tau, amyloid beta peptide, SOD1, C9orf72, and TDP-43; see, e.g.,Blokhuis et al., Acta Neuropathol. 125 (2013), 777-794. Human-derivedmonoclonal antibodies against the mentioned proteins are known in theart; see, e.g., international application WO2008/081008 for anti-abetaantibody, WO2010/069603 for anti-α-synuclein antibody; WO2012/049570 foranti-tau antibody; WO2012/080518 for anti-SOD1 antibody; WO2012/113775for anti-ankyrin antibody; WO2013/061163 for anti-TDP-43 antibody andEuropean patent application EP 14 187 180.6 and its subsequentinternational application for C9orf72. Bi- and multispecific antibodiescan be generated by methods well known in the art, for example bychemical recombination of monoclonal immunoglobulin Gi fragments asdescribed, e.g., by Brennan et al., Science. 229 (1985), 81-83, orrecombinant simultaneously co-expression of the appropriate heavy andlight chain and corresponding pairing; see, e.g., Lewis et al., NatureBiotechnology 32 (2014), 191-198; for review see, e.g., Kontermann, mAbs4 (2012), 182-197 and Kontermann and Brinkmann, Drug Discovery Today 20(2015), 838-847.

Alternatively, or in addition the bi- or multi-specific antibodycomprises at least a first and second antigen-binding site, i.e.variable domain specific for two distinct epitopes of HTT, preferablywherein one or both variable regions are derived from any one of thesubject antibodies illustrated in the appended Examples and Figures, andas further described herein. Thus, in a preferred embodiment thebispecific antibody of the present invention comprises two bindingsites/domains of an antibody which recognizes a polyP-region, apolyQ/polyP-region, the P-rich-region, the C terminal-region, theN-terminal region or a conformational epitope of HTT exon 1 protein. Theepitopes of the subject antibodies illustrated in the Examples aresummarized in FIG. 20. Accordingly, in one embodiment the bispecificantibody of the present invention recognizes at least two differentepitopes depicted in FIG. 20 and has the combined binding specificitiesof the cognate subject antibody, respectively.

The antigen-binding fragment of any one the subject antibodies disclosedherein can be a single chain Fv fragment, an F(ab′) fragment, an F(ab)fragment, and an F(ab′)₂ fragment, or any other antigen-bindingfragment. However, as mentioned in a particularly preferred embodiment,the antibody or HTT-binding fragment, synthetic or biotechnologicalderivative thereof is a human IgG isotype antibody and comprises atleast part of the constant region. Alternatively, the antibody is achimeric human-rodent or rodentized antibody such as murine ormurinized, rat or ratinized antibody, the rodent versions beingparticularly useful for diagnostic methods and studies in animals.

Furthermore, the present invention relates to compositions comprisingthe antibody of the present invention or antigen-binding fragment,synthetic or biotechnological derivative thereof and toimmunotherapeutic and immunodiagnostic methods using such compositionsin the prevention, diagnosis or treatment of diseases and/or disordersassociated with HTT amyloidosis, wherein an effective amount of thecomposition is administered to a patient in need thereof.

The present invention also relates to polynucleotides encoding at leasta variable region of an immunoglobulin chain of the antibody of theinvention. Preferably, said variable region comprises at least onecomplementarity determining region (CDR) of the V_(H) and/or V_(L) ofthe variable region as set forth in FIG. 1. Preferably, thepolynucleotide is a cDNA.

Accordingly, the present invention also encompasses vectors comprisingsaid polynucleotides and host cells transformed therewith as well astheir use for the production of an antibody and equivalent bindingmolecules which are specific for HTT and preferably are capable ofbinding mutated and/or aggregated HTT species or fragments thereof.Means and methods for the recombinant production of antibodies andmimics thereof as well as methods of screening for competing bindingmolecules, which may or may not be antibodies, are known in the art.

However, as described herein, in particular with respect to therapeuticapplications in human the antibody of the present invention is a humanantibody in the sense that application of said antibody is substantiallyfree of an immune response directed against such antibody otherwiseobserved for chimeric and even humanized antibodies. Hence, the presentinvention also relates to the use of the cDNA, vector and host celldescribed herein and illustrated in the Examples for the production ofan anti-HTT antibody, in particular human-derived anti-HTT antibody or abiotechnological derivative thereof.

Furthermore, disclosed herein are compositions and methods that can beused to identify HTT, in particular mutated and/or aggregated HTTspecies or fragments in vitro, e.g. in samples and/or in vivo. Thedisclosed anti-HTT antibodies and binding fragments thereof can be usedto screen human blood, plasma, serum, saliva, peritoneal fluid,cerebrospinal fluid (“CSF”), and urine for the presence of HTT and/ormutated and/or aggregated HTT species or fragments thereof in samples,for example, by using ELISA-based or surface adapted assay. In oneembodiment the present invention relates to a method of diagnosing ormonitoring the progression of a disease and/or disorder related tomutated and/or aggregated HTT species or fragments thereof in a subject,the method comprising determining the presence of mutated, and/oraggregated HTT species or fragments in a sample from the subject to bediagnosed with at least one antibody of the present invention or anHTT-binding molecule and/or binding molecules for mutated and/oraggregated HTT species or fragments having substantially the samebinding specificities of any one thereof, wherein the presence ofmutated and/or aggregated HTT species or fragments is indicative of thedisorder.

Accordingly, the present invention also relates to a method of preparinga pharmaceutical composition for use in the treatment of a disorderassociated with or caused by HTT aggregates, the method comprising:

-   -   (a) expressing the cDNA of the present invention and/or        culturing the host cell of the present invention under        appropriate culture conditions suitable for the production of        the anti-HTT antibody, in particular human-derived anti-HTT        antibody or a biotechnological derivative thereof;    -   (b) purifying the antibody, biotechnological derivative or        immunoglobulin chain(s) thereof from a reaction mixture and the        culture, respectively, to pharmaceutical grade; and    -   (c) admixing the antibody or biotechnological derivative thereof        with a pharmaceutically acceptable carrier.

Furthermore, in one embodiment of the present invention the anti-HTTantibodies and HTT-binding molecules comprising at least one CDR of anantibody of the present invention are provided for the preparation of acomposition for in vivo detection (also called in vivo imaging) of ortargeting a therapeutic and/or diagnostic agent to HTT, in particularmutated and/or aggregated HTT species or fragments in the human oranimal body. The methods and compositions disclosed herein can aid indiseases and/or disorders associated with HTT aggregation or amyloidosisand characterized, e.g., by the occurrence of aggregated forms of HTTand can be used to monitor disease progression and therapeutic efficacyof the therapy provided to the subject, for example in in vivo imagingrelated diagnostic methods. In one embodiment the in vivo detection(imaging) comprises scintigraphy, positron emission tomography (PET),single photon emission tomography (SPECT), near infrared (NIR) opticalimaging or magnetic resonance imaging (MRI).

Hence, it is a particular object of the present invention to providemethods for treating, diagnosing or preventing a disease and/or disorderassociated with HTT amyloidosis. The methods comprise administering aneffective concentration of a preferably human antibody or antibodyderivative to the subject where the antibody targets HTT or fragmentsthereof, preferably mutated and/or aggregated or misfolded HTT speciesor fragments thereof.

In a further aspect the present invention provides a peptide having anepitope of HTT, preferably of mutated and/or aggregated HTT species orfragments thereof specifically recognized by an antibody of the presentinvention. Said peptide comprises or consists of an amino acid sequenceas indicated below in the detailed description and in the Examples or amodified sequence thereof in which one or more amino acids aresubstituted, deleted and/or added. Additionally, the present inventionprovides a method for diagnosing diseases and/or disorders associatedwith HTT amyloidosis in a subject, comprising a step of determining thepresence of an antibody that binds to said peptide in a biologicalsample of said subject.

Further embodiments of the present invention will be apparent from thedescription and Examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid sequences of the variable regions of human antibodiesNI-302.33C11, NI-302.63F3, NI-302.35C1, NI-302.31F11, NI-302.2A2,NI-302.6N9, NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4,NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8,NI-302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1,NI-302.8F1, NI-302.52C9, NI-302.46C9, NI-302.15E8, NI-302.15D3,NI-302.64E5, NI-302.7D8, NI-302.72F10, NI-302.12H2, NI-302.8M1 andNI-302.4A6. Framework (FR) and complementarity determining regions(CDRs) are indicated with the CDRs being underlined. The Kabat numberingscheme was used (cf. www.bioinf.org.uk/abs/).

FIG. 2: Characterization of Huntingtin (HTT) exon 1 proteins andaggregates. (A) Cloning of GST-HttEx1Q21 (GST-HD21), GST-HttEx1Q35(GST-HD35) and GST-HttEx1Q49 (GST-HD49) expression constructs; (B)Coomassie dye staining upon SDS-PAGE of purified GST only (lane 1),GST-HttExon1Q21 (GST-HD21, lane 2), GST-HttExon1Q35 (GST-HD35, lane 3)and GST-HttExon1Q49 (GST-HD49, lane 4) proteins showing good purity butalso some additional bands; (C) Characterization of in vitro HD21, HD35and HD49 time-resolved in vitro aggregation reactions by dot-blot (left)and filter retardation analysis (right) with polyclonal HD-1 antibody asdetection antibody. Aggregation reactions of HD35 at 24 hours or HD49reactions after 3 hours show aggregates larger than the pore size of 0.2μm detectable by HD-1 in the filter retardation assay analysis; (D)Characterization of in vitro HD35 and HD49 preparations by electronmicroscopy. Aggregation reactions of HD35 after 24 hours [A, E] or HD49reactions after 1 hour [B, F], 3 hours [C, G] or 24 hours [D, H].Overview pictures [A-D] with 1,000× magnification and detailedstructures [E-H] at 66,000× magnification.

FIG. 3: Characterization of the binding affinity of anti-polyPdomain-binding antibody NI-302.33C11. (A) NI-302.33C11 binding affinityfor different HTT species determined by direct ELISA; (B) NI-302.33C11EC₅₀ determinations for aggregated HD49 (●), aggregated HD21 (▪),soluble GST-HD49 (▴) and GST-HD21 (▾) Htt Exon 1 proteins using directELISA. NI-302.33C11 antibody binds with similar EC₅₀ values to all fourspecies; and (C) NI-302.33C11 binding analysis to HTT aggregates on invitro HD21, HD35 and HD49 time-resolved in vitro aggregation reactionsby dot-blot (left) and filter retardation assay (right) withpreferential binding to later (aggregated) reactions of HD35 and HD49 inthe dot-blot assays and aggregates of HD35 and HD49 in the filterretardation assay.

FIG. 4: Determination of NI-302.33C11 antibody binding epitope by scanof overlapping peptides. At the top: pepscan image after NI-302.33C11antibody hybridization. Below: graphical overviews of peptides sequencesbound by NI-302.33C11 antibody. Overlapping amino acids between peptides(putative binding epitope) being recognized by the NI-302.33C11 antibodyare highlighted in bold in the consensus sequences. The HRP-conjugateddonkey anti-human IgG Fcγ detection antibody alone does not bind anylinear huntingtin peptide.

FIG. 5: NI-302.33C11 binds to the polyP-domain of HTT. EC₅₀determinations for GST-HD49 (●), BSA-coupled P-rich domain peptide (♦),BSA-coupled C-terminal peptide (▪) or BSA-coupled polyP peptide (▴)using direct ELISA.

FIG. 6: Characterization of the purity and integrity as well as thebinding specificity of NI-302.33C11 antibody. SDS-PAGE analysis followedby Coomassie staining of 2 and 10 μg recombinant human NI-302.33C11anti-polyP domain antibody.

FIG. 7: Characterization of binding affinity of anti-proline-rich domainantibody NI302.63F3. (A) NI-302.63F3 binding affinity for different HTTspecies determined by direct ELISA; (B) NI-302.63F3 EC₅₀ determinationsfor aggregated HD49 (●), aggregated HD21 (▪), soluble GST-HD49 (▴) andGST-HD21 (▾) Htt Exon 1 proteins using direct ELISA. NI-302.63F3antibody has a similar EC₅₀ values to all four species; (C)Characterization of antibody NI-302.63F3 on in vitro HD21, HD35 and HD49time-resolved in vitro aggregation reactions by dot-blot (left) andfilter retardation assay (right) with preferential binding to huntingtinwith expanded polyQ tracts (HD49>HD35) in the dot-blot assays andaggregates of HD35 and HD49 in the filter retardation assay.

FIG. 8: Determination of NI-302.63F3 antibody binding epitope by scan ofoverlapping peptides. At the top: pepscan image after NI-302.63F3antibody hybridization. Below: graphical overviews of peptides sequencesbound by NI-302.63F3 antibody. Overlapping amino acids between peptides(putative binding epitope) being recognized by the NI-302.63F3 antibodyare highlighted in bold in the consensus sequences. The HRP-conjugateddonkey anti-human IgG Fcγ detection antibody alone does not bind anylinear huntingtin peptide.

FIG. 9: NI-302.63F3 binds to the P-rich domain of HTT. EC₅₀determinations for GST-HD49 (●), BSA-coupled P-rich domain peptide (♦),BSA-coupled C-terminal peptide (▪) or BSA-coupled polyP peptide (▴)using direct ELISA.

FIG. 10: Characterization of the purity and integrity as well as thebinding specificity of NI-302.63F3 antibody. SDS-PAGE analysis followedby Coomassie staining of 2 and g recombinant human NI-302.63F3anti-proline-rich domain antibody.

FIG. 11: Characterization of the binding affinity of anti-C-terminaldomain-binding antibody NI 302.35C1. (A) NI-302.35C1 binding affinityfor different HTT species determined by direct ELISA; (B) NI-302.35C1EC₅₀ determinations for aggregated HD49 (●), aggregated HD21 (▪),soluble GST-HD49 (▴) and GST-HD21 (▾) Htt Exon 1 proteins using directELISA; (C) Characterization of antibody NI-302.35C1 on in vitro HD21,HD35 and HD49 time-resolved in vitro aggregation reactions by dot-blot(left) and filter retardation assay (right) with preferential binding tolater (aggregated) reactions of HD35 and HD49 in the dot-blot assays andaggregates of HD35 and HD49 in the filter retardation assay.

FIG. 12: NI-302.35C1 binds to the BSA-coupled C-terminal domain peptideof HTT. EC50 determinations for GST-HD49 (●), BSA-coupled P-rich domainpeptide (♦), BSA-coupled C-terminal peptide (▪) or BSA-coupled polyPpeptide (▴) using direct ELISA.

FIG. 13: Characterization of the purity and integrity as well as thebinding specificity of NI-302.35C1 antibody. SDS-PAGE analysis followedby Coomassie staining of 2 and g recombinant human NI-302anti-C-terminal domain antibody.

FIG. 14: Characterization of binding affinity of anti-N-terminal domainantibody NI302.15E8. (A) NI-302. 15E8 binding affinity for different HTTspecies determined by direct ELISA; (B) NI-302.15E8 EC₅₀ determinationsfor aggregated HD49 (●), aggregated HD21 (▪), soluble GST-HD49 (▴) andGST-HD21 (▾) Htt Exon 1 proteins using direct ELISA. NI-302. 15E8antibody has a higher affinity binding EC₅₀ values to non-aggregatedspecies.

FIG. 15: NI-302.15E8 binds to the BSA-coupled N-terminal domain peptideof HTT. EC₅₀ determinations for GST-HD49 (●), BSA-coupled N-terminalpeptide (▾) BSA-coupled P-rich domain peptide (♦), BSA-coupledC-terminal peptide (▪) or BSA-coupled polyP peptide (▴) using directELISA.

FIG. 16: Target specificity analysis by direct ELISA. NI-302 antibodies(A) NI-302.33C11, (B) NT-302.63F3, and (C) NT-302.35C1 and (D)NT-302.15E8 do not bind unrelated aggregating protein targets as shownin the binding specificity analysis by direct ELISA.

FIG. 17: Spine density is significantly reduced in hippocampal slicecultures of Tg(HDexon1)62Gpb/1J transgenic mice compared tonon-transgenic littermates. (A-D) Overview of GFP positive hippocampalneurons of non-transgenic littermates (A, C) vs. Tg(HDexon1)62Gpb/1Jmice (B, D), showing a single dendrite with the individual spines athigher magnification (C, D). (E) Significant reduction of dendriticspine density in transgenic vs. wildtype animals (n=3-7 slices per groupfrom 2 wt or 3 transgenic animals). (F) Attenuation of dendritic spinedensity loss by antibodies NI-302.11F11 and 302.63F3 in slices oftransgenic mice. (n=8-13 slices per group from a total of 12 transgenicanimals). Data represent the mean±SEM. *p<0.05 (MWU), #p=0.05.

FIG. 18: Penetration of NI-302 antibodies in the brain of R6/1 animalmodel. (A) Mean NI-302.31F11 (●) and NI-302.35C1 (▪) plasma and braindrug levels in R6/1 transgenic animals after a single intraperitonealinjection of 50 mg/kg. Data represent the mean±SEM. n=3 for each group;(B) Plasma and brain drug levels of individual mice after a single doseof 50 mg/kg.

FIG. 19: EC₅₀ determinations of human-derived HTT antibodies foraggregated HD49 (●), aggregated HD21 (▪), soluble GST-HD49 (▴) andGST-HD21 (▾) Htt Exon 1 proteins using direct ELISA. (A): antibodyNI-302.31F11; (B): antibody NI-302.6N9; (C): antibody NI-302.74C11; (D):antibody NI-302.15C9; (E): antibody NI-302.39C12; (F): antibodyNI-302.11A4; (G): antibody NI-302.22H9; (H): antibody NI-302.44D7; (I):antibody NI-302.37C12; (J): antibody NI-302.55D8; (K): antibodyNI-302.7A8; (L): antibody NI-302.78H12; (M): antibody NI-302.71F6; (N):antibody NI-302.11H6; (O): antibody NI-302.3D8; (P): antibodyNI-302.46C9; (Q): antibody NI-302.2A2; (R): antibody NI-302.15D3. Someantibodies (e.g. NI-302.37C12 (I), NI-302.55D8 (J), NI-302.11A4 (F) orNI-302.22H9 (G)) seem to have preferred binding to uncut GST-HTT proteinsuggesting that these antibodies preferentially recognize uncut solubleGST-HD constructs whereas some antibodies (e.g. NI-302.74C11 (C) orNI-302.71F6 (M)) showed high affinity binding with similar EC-values toall HTT preparation suggesting that they bind to an epitope that issimilar exposed in aggregated and uncut HTT exon 1 constructs in theELISA assay.

FIG. 20: Characterization of binding affinity by direct ELISA. Bindingaffinity to the different HTT proteins of human-derived HTT-specificantibodies

FIG. 21: Characterization of antibody NI-302.44D7, NI-302.37C12,NI-302.15F9 and NI-302.71F6 on in vitro HD21, HD35 and HD49time-resolved in vitro aggregation reactions by dot-blot (left) andfilter retardation assay (right) with preferential binding in particularNI-302.15F9 and NI-302.71F6 to later (aggregated) reactions of HD35 andHD49 in the dot-blot assays and SDS stable aggregates of HD35 and HD49in the filter retardation assay.

FIG. 22: Target specificity analysis by direct ELISA. NI-302 antibodies(A) NI-302.31F11, (B) NI-302.6N9, (C) NI-302.46C9, (D) NI-302.8F1, (E)NI-302.2A2, (F) NI-302.74C11, (G) NI-302.15F9, (H) NI-302.39G12, (I)NI-302.11A4, (J) NI-302.22H9, (K) NI-302.44D7, (L) NI-302.55D8, (M)NI-302.7A8, (N) NI-302.78H12, (O) NI-302.71F6, (P) NI-302.11H6, and (Q)NI-302.3D8 do not bind unrelated aggregating protein targets as shown inthe binding specificity analysis by direct ELISA

FIG. 23: Determination of NI-302 antibody binding epitope by scan ofoverlapping peptides. At the top: pepscan image after NI-302 antibodyhybridization. Below: graphical overviews of peptides sequences andNI-302 antibody binding score to the single peptides are shown.Overlapping amino acids between peptides (putative binding epitope)being recognized by the NI-302 antibody are highlighted in gray in theconsensus sequences. The HRP-conjugated donkey anti-human IgG Fcγdetection antibody alone does not bind any linear huntingtin peptide.(A) NI-302.31F11 1 μg/ml on a 21 spot membrane, (B) NI-302.74C11 1 μg/mlon a 16 spot membrane, (C) NI-302.15F9 1 μg/ml on a 16 spot membrane,(D) NI-302.39G12 1 μg/ml on a 16 spot membrane, (E) NI-302.11A4 1 μg/mlon a 16 spot membrane, (F) NI-302.22H9 1 μg/ml on a 16 spot membrane,(G) NI-302.44D7 1 μg/ml on a 16 spot membrane, (H) NI-302.37C12 1 μg/mlon a 16 spot membrane, (I) NI-302.55D8 1 μg/ml on a 16 spot membrane,(J) NI-302.7A8 1 μg/ml on a 21 spot membrane, (K) NI-302.78H12 1 μg/mlon a 16 spot membrane, (L) NT-302.71F6 1 μg/ml on a 16 spot membrane,(M) NI-302.11H6 1 μg/ml on a 21 spot membrane, (N) NI-302.18A1 1 μg/mlon a 21 spot membrane, (O) NI-302.3D8 1 μg/ml on a 21 spot membrane, (P)NI-302.46C9 1 μg/ml on a 21 spot membrane and (Q) NI-302.52C9 1 μg/ml ona 21 spot membrane, (R) NI-302.2A2 1 μg/ml on a 21 spot membrane, (S)NI-302.15E8 1 μg/ml on a 21 spot membrane and (T) NI-302.15D3 1 μg/ml ona 21 spot membrane.

FIG. 24: Immunohistochemical analysis of NI-302 antibodies revealsprominent staining of neuronal intranuclear inclusions in striatalneurons of late disease stage Tg(HDexon1)62Gpb/1J transgenic animals at5 nM (74C11, 39C12, 11A4, 22H9, 78H12, 37C12, 7D8, 72F10), or 50 nMconcentrations (15F9, 71F6, 55D8, 44D7, 7A8, 64E5). Mab5492 is acommercially available N-terminal HTT antibody.

FIG. 25: Basic characterization of R6/1 transgenic mouse modelTg(HDexon1)61Gpb/J. (A) Survival curve, (B) body weight curve and (C)total brain wet weight during the disease progression of this animalmodel. (D-H) Characterization of appearance of neuronal intranuclearinclusions with disease progression in the striatum by staining withNI-302.33C11 HTT antibody.

FIG. 26: Basic characterization of B6C3-Tg(HD82Gln)81Dbo/J (N171-82Q)transgenic mouse model. (A) Survival curve, (B) body weight curve duringthe disease progression and (C) total brain wet weight at end stage ofthis animal model. (D-F) Characterization of appearance of neuronalintranuclear inclusions with disease progression in the striatum bystaining with Mab5492 HTT antibody.

FIG. 27: Immunohistochemical analysis with 50 nM ofNI-302.33C11(polyP-epitope) shows staining of neuronal intranuclearinclusions in cortical neurons of four different Huntington Diseasepatients (A-D) and in striatal neurons of 270 day old, late diseasestage B6.Cg-Tg(HDexon1)61Gpb/J) transgenic animals at 1 (E) and 5 nM (F)concentration. No staining is detected in non-transgenic littermates(G), if primary antibody is omitted during the staining (H) or if tissueof non-Huntington Disease controls is stained with 50 nM ofNI-302.33C11.

FIG. 28: Immunohistochemical analysis with 50 nM of NI-302.63F3 (P-richdomain epitope) shows staining of neuronal intranuclear inclusions (A-C)and staining of some neurites (D) of cortical neurons of four differentHuntington Disease patients (A-D) and in striatal neurons of 270 dayold, late disease stage B6.Cg-Tg(HDexon1)61Gpb/J) transgenic animals at1 (E) and 50 nM (F) concentration. No staining is detected innon-transgenic littermates (G), if primary antibody is omitted duringthe staining (H) or if tissue of non-Huntington Disease controls isstained with 50 nM of NI-302.63F3.

FIG. 29: Immunohistochemical analysis with 100 nM of NI-302.35C1 (endExon 1 epitope) shows staining of neuronal intranuclear inclusions (A-C)and staining of some neurites (D) of cortical neurons of four differentHuntington Disease patients (A-D) and in striatal neurons of 270 dayold, late disease stage B6.Cg-Tg(HDexon1)61Gpb/J) transgenic animals at1 (E) and 50 nM (F) concentration. No staining is detected innon-transgenic littermates (G), if primary antibody is omitted duringthe staining (H) or if tissue of non-Huntington Disease controls isstained with 100 nM of NI-302.35C1.

FIG. 30: Immunohistochemical analysis with commercially availableanti-polyQ antibody Mab1574 (1:2000, Chemicon) shows staining ofneuronal intranuclear and cytoplasmic inclusions and staining of someneurites (A, D) of cortical neurons of four different Huntington Diseasepatients (A-D) and in striatal neurons of presymptomatic, 150 day old(E) and 270 day old (F), late disease stage B6.Cg-Tg(HDexon1)61Gpb/J)transgenic animals. No staining is detected in non-transgeniclittermates (G), if primary antibody is omitted during the staining (H)or if tissue of non-Huntington Disease controls is stained with Mab1574.

FIG. 31: EC₅₀ determinations of human-derived HTT antibodies foraggregated HD49 (●), aggregated HD21 (▪), soluble GST-HD49 (▴) andGST-HD21 (▾) Htt Exon 1 proteins using direct ELISA. Some antibodies(e.g. NI-302.64E5 (A) or NI-302.7D8 (B)) seem to have preferred bindingto uncut GST-HD49 protein suggesting that these antibodiespreferentially recognize uncut soluble GST-HD constructs containinglonger polyQ repeats. Antibody NI-302.72F10 (C) shows preference to HD21constructs and some antibodies (e.g. NI-302.4A6 (D), NI-302.12H2 (E) orNI-302.8M1 (F)) showed high affinity binding with similar EC-values toall HTT preparation suggesting that they bind to an epitope that issimilar exposed in aggregated and uncut HTT exon 1 constructs in theELISA assay.

FIG. 32: Characterization of antibody (A) NI-302.64E5, (B) NI-302.7D8,(C) NI-302.72F10, (D) NI-302.4A6, (E) NI-30212H2, (F) NI-302.8M1 and (G)NI-302.33C11 (as control) on in vitro HD21, HD35 and HD49 time-resolvedin vitro aggregation reactions by dot-blot (left) and filter retardationassay (right) with preferential binding in particular of NI-302.64E5 andNI-302.72F10 to later (aggregated) reactions of HD35 and/or HD49 in thedot-blot assays and SDS stable aggregates of HD35 and HD49 in the filterretardation assay.

FIG. 33: Target specificity analysis by direct ELISA. NI-302 antibodies(A) NI-302.64E5, (B) NI-302.7D8, (C) NI-302.72F10, (E) NI-302.12H2 and(F) NI-302.8M1 do not bind unrelated aggregating protein targets asshown in the binding specificity analysis by direct ELISA, except (D)NI-302.4A6 which shows some binding to p53.

FIG. 34: Study of C-terminal domain-binding antibody NI 302.35C1 onbehavioral performance during task-specific training and sensorimotorability in a mice model of HD. (A) The plus-maze analysis was used toinvestigate the level of anxiety in the R6/1 mice. At 6 months of age,NI-302.35C1 treated R6/1 animals spend less time in the open arms,entered the open arms less frequently and did less unprotected head dipson the open arm compared to vehicle treated R6/1 animals. Hence theNI-302.35C1 treated R6/1 mice displayed a more anxious phenotype,comparable to the non-transgenic littermates. (B) NI-302.35C1 treatedR6/1 animal showed an improved performance in the pole test compared tovehicle treated R6/1 animals reaching levels similar to non-transgenicanimals.

FIG. 35 Determination of NI-302 antibody binding epitope by scan ofoverlapping peptides. At the top: pepscan image after NI-302 antibodyhybridization. Below: graphical overviews of peptides sequences areshown. Overlapping amino acids between peptides (putative bindingepitope) being recognized by the NI-302 antibody are shown in theconsensus sequence below. The HRP-conjugated donkey anti-human IgG Fcγdetection antibody alone does not bind any linear huntingtin peptide.(A) NI-302.64E5, (B) NI-302.7D8, (C) NI-302.72F10, (D) NI-302.4A6, (E)NI-302.12H2, (F) NT-302.8M1 all antibodies at 1 μg/ml on the 21 spotmembrane.

FIG. 36 Amino acid sequence alignment of the CDRs in the V_(H) and V_(L)or V_(K) chains of NI-302 antibodies. Each sequence was checked in termsof conserved amino acids, segments, or other motifs revealing anaccumulation of tyrosines in the CDRs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to immunotherapy andnon-invasive methods for the detection of diseases and/or disorders aswell as conditions associated with the presence of pathologic, oftenmutant and/or aggregated forms of huntingtin (HTT). More specifically,the present invention relates to recombinant human-derived monoclonalantibodies and HTT-binding fragments, synthetic and biotechnologicalderivatives thereof, which have been generated based on sequenceinformation obtained from selected human donor populations and arecapable of binding to such HTT isoforms and antigens thereof. Therecombinant human-derived monoclonal antibody of the present inventionis advantageously characterized by specifically binding to mutatedand/or aggregated HTT species and/or fragments thereof allowing atargeting for treatment and/or diagnosis of pathological altered HTTspecies. Due to their human derivation, the resulting recombinantantibodies of the present invention can be reasonably expected to beefficacious and safe as therapeutic agent, and highly specific as adiagnostic reagent for the detection of pathological HTT without givingfalse positives.

In addition, the present invention relates to the human monoclonalantibody and any derivatives thereof described herein for use in thetreatment of patients either alone or with other agents utilized forsymptoms associated with HTT amyloidosis, wherein the antibody of thepresent invention and any of its derivatives is designed to beadministered concomitantly with the agent suppressing side effects orsequentially before or after administration of the same. In thiscontext, the anti-HTT antibody and HTT-binding fragment of the presentinvention are preferably substantially non-immunogenic in human. In oneembodiment of the present invention, pharmaceutical compositions areprovided comprising both a human monoclonal antibody of the presentinvention or any derivatives thereof and one or more drug utilized forsymptoms associated with HTT amyloidosis.

I. Definitions

Unless otherwise stated, a term as used herein is given the definitionas provided in the Oxford Dictionary of Biochemistry and MolecularBiology, Oxford University Press, 1997, revised 2000 and reprinted 2003,ISBN 0 19 850673 2.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

Huntingtin (HTT), also known as IT15 is a disease gene linked toHuntington's disease (HD), a neurodegenerative disorder characterized byloss of striatal neurons. It is thought that HD is caused by anexpanded, unstable trinucleotide repeat in the HTT gene, whichtranslates as a polyglutamine repeat in the protein product. A fairlybroad range in the number of trinucleotide repeats has been identifiedin normal controls, and repeat numbers in excess of 35-40 have beendescribed as pathological. The HTT locus (NG_009378.1; 4830 to 174286;NCBI RefSeqGene) is large, spanning 180 kb and consisting of 67 exons.

In this context, it has been demonstrated that an N-terminal fragment ofmutant HTT, i.e. exon 1 protein of the HTT gene, with an expanded CAGrepeat represents the “toxic” species of HTT which is sufficient tocause aggregation and a progressive neurological phenotype in transgenicmice; see, e.g., Mangiarini et al., Cell 87 (1996), 493-506 and Ross etal., Lancet Neurol. 10 (2011), 83-98, DiFiglia et al, Science 277(1997), 1990-1993, Gutekunst et al., J Neurosci 19(7) (1999), 2522-2534.

Unless stated otherwise, by “specifically recognizing HTT”, “antibodyspecific to/for HTT” and “anti-HTT antibody” antibodies are meant whichspecifically, generally, and collectively bind to HTT, wherein HTT referto different forms the HTT including but not limited to the native formof HTT as well as other forms of HTT, e.g. pathologically altered HTT,such as mutated, misfolded and/or aggregated HTT. Provided herein arehuman-derived antibodies selective for full-length and/or fragmentsand/or mutated, misfolded and/or aggregated forms of HTT.

If not specifically indicated otherwise, the term “HTT”, is usedinterchangeably to specifically refer to the different forms ofhuntingtin (HTT). The term “HTT” is also used to generally identifyother conformers of HTT, for example, pathologically altered forms ofHTT such as misfolded and/or aggregated forms of HTT. Furthermore,unless specifically indicated otherwise the term HTT in particular meansHTT exon1 and the soluble HTT refers to the corresponding GST-fusionproteins. The term “HTT” is also used to refer collectively to all typesand forms of HTT, such as mutated HTT. Added letters in front of theterms, e.g. HTT, are used to indicate the organism the particularortholog is originating from, e.g. hHTT for human HTT or mHTT for murineorigin.

The anti-HTT antibodies disclosed herein specifically bind HTT andepitopes thereof and to various conformations of HTT and epitopesthereof. For example, disclosed herein are antibodies that specificallybind pathologically altered HTT species or fragments thereof, such asmutated, misfolded, and/or aggregated forms of HTT or fragments thereof.The term (pathologically) mutated, misfolded, and/oraggregated/aggregates of HTT is used interchangeable to specificallyrefer to the aforementioned forms. The term (pathological) “aggregatedforms” or “aggregates” as used herein describes the products of anaccumulation or cluster formation due to HTT erroneous/pathologicalinteraction with one another. These aggregates, accumulations or clusterforms may be, substantially consist or consist of both HTT and/or HTTfragments and of non-fibrillar oligomers and/or fibrillar oligomers andfibrils thereof. As used herein, reference to an antibody that“specifically binds”, “selectively binds”, or “preferentially binds” HTTrefers to an antibody that does not bind other unrelated proteins.

In one example, a HTT antibody disclosed herein can bind HTT or anepitope thereof and show no binding above about 2 times background forother proteins. In a preferred embodiment, the antibody of the presentinvention does not substantially recognize unrelated amyloid-formingproteins selected from the group consisting of paired helical filament(PHF)-tau, TAU, alpha-synuclein, transactive response DNA bindingprotein 43 (TDP43), islet amyloid polypeptide (IAPP), transthyrethin(TTR), serum amyloid A (SAA); see Examples 8, 13, 18 and 31. An antibodythat “specifically binds” or “selectively binds” a HTT conformer refersto an antibody that does not bind all conformations of HTT, i.e., doesnot bind at least one other HTT conformer. For example, disclosed hereinare antibodies that can preferentially bind to mutated and/or aggregatedforms of HTT both in vitro and in tissues obtained from patients withdiseases and/or disorders associated with HTT amyloidosis or with a riskto develop diseases and/or disorders associated with HTT amyloidosis. Inanother embodiment of the present invention the antibodies of thepresent invention specifically targets different regions of the HTT exon1; see, e.g., FIGS. 5, 9, 12, 14, 15. Since the anti-HTT antibodies ofthe present invention have been isolated from human subjects, they mayalso be called “human auto-antibodies” or “human-derived antibodies” inorder to emphasize that those antibodies were indeed expressed initiallyby the subjects and are not synthetic constructs generated, for example,by means of human immunoglobulin expressing phage libraries orxenogeneic antibodies generated in a transgenic animal expressing partof the human immunoglobulin repertoire which hitherto represented onecommon method for trying to provide human-like antibodies. On the otherhand, the human-derived antibody of the present invention may be denotedsynthetic, recombinant, and/or biotechnological in order distinguish itfrom human serum antibodies per se, which may be purified via protein Aor affinity column.

However, a particular advantage of the therapeutic approach of thepresent invention lies in the fact that the antibodies of the presentinvention are derived from B cells or B memory cells from healthy humansubjects with no signs of a disease showing the occurrence of, orrelated to misfolded/aggregated HTT and thus are, with a certainprobability, capable of preventing a clinically manifest disease relatedto misfolded/aggregated HTT, or of diminishing the risk of theoccurrence of the clinically manifest disease, or of delaying the onsetor progression of the clinically manifest disease. Typically, theantibodies of the present invention also have already successfully gonethrough somatic maturation, i.e. the optimization with respect toselectivity and effectiveness in the high affinity binding to the targetHTT molecules by means of somatic variation of the variable regions ofthe antibody. The knowledge that such cells in vivo, e.g. in a human,have not been activated by means of related or other physiologicalproteins or cell structures in the sense of an autoimmunological orallergic reaction is also of great medical importance since thissignifies a considerably increased chance of successfully living throughthe clinical test phases. So to speak, efficiency, acceptability andtolerability have already been demonstrated before the preclinical andclinical development of the prophylactic or therapeutic antibody in atleast one human subject. It can thus be expected that the human anti-HTTantibodies of the present invention, both its target structure-specificefficiency as therapeutic agent and its decreased probability of sideeffects significantly increase its clinical probability of success.

In contrast, antibodies derived from cDNA library's or phage displaysare artificial molecules such as a humanized antibody which is still ofmurine origin and thus foreign to the human body. Therefore the clinicalutility and efficacy of the therapeutic antibodies can be limited by theproduction of anti-drug antibodies (ADAs), which can influence theefficacy and pharmacokinetics of the antibodies and sometimes lead toserious side effects, see e.g. Igawa et al., MAbs. 3 (2011), 243-252. Inparticular, humanized antibodies or antibodies generated with recenthuman-antibody-generation technologies are in contrast to thehuman-derived antibodies such as those of the present invention prone toinduce an antibody response and these human-like antibodies derived frome.g. phage display such as adalimumab have been reported to induce ADAproduction, see, e.g., Mansour, Br. J. Ophthalmol 91 (2007), 274-276 andIgawa et al., MAbs. 3 (2011), 243-252. Therefore, human-derivedantibodies which are not prone to undesired immune response are morebeneficial for the patient than artificial molecules derived fromlibraries or displays.

The term “peptide” is understood to include the terms “polypeptide” and“protein” (which, at times, may be used interchangeably herein) withinits meaning. Similarly, fragments of proteins and polypeptides are alsocontemplated and may be referred to herein as “peptides”. Nevertheless,the term “peptide” preferably denotes an amino acid polymer including atleast 5 contiguous amino acids, preferably at least 10 contiguous aminoacids, more preferably at least 15 contiguous amino acids, still morepreferably at least 20 contiguous amino acids, and particularlypreferred at least 25 contiguous amino acids. In addition, the peptidein accordance with present invention typically has no more than 100contiguous amino acids, preferably less than 80 contiguous amino acidsand more preferably less than 50 contiguous amino acids.

Polypeptides:

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, “peptides,” “dipeptides,”“tripeptides, “oligopeptides,” “protein,” “amino acid chain,” or anyother term used to refer to a chain or chains of two or more aminoacids, are included within the definition of “polypeptide,” and the term“polypeptide” may be used instead of, or interchangeably with any ofthese terms.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation andderivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Recombinant peptides, polypeptides or proteins” refer to peptides,polypeptides or proteins produced by recombinant DNA techniques, i.e.produced from cells, microbial or mammalian, transformed by an exogenousrecombinant DNA expression construct encoding the fusion proteinincluding the desired peptide. Proteins or peptides expressed in mostbacterial cultures will typically be free of glycan. Proteins orpolypeptides expressed in yeast may have a glycosylation patterndifferent from that expressed in mammalian cells.

Included as polypeptides of the present invention are fragments,derivatives, analogs or variants of the foregoing polypeptides and anycombinations thereof as well. The terms “fragment,” “variant,”“derivative”, and “analog” include peptides and polypeptides having anamino acid sequence sufficiently similar to the amino acid sequence ofthe natural peptide. The term “sufficiently similar” means a first aminoacid sequence that contains a sufficient or minimum number of identicalor equivalent amino acid residues relative to a second amino acidsequence such that the first and second amino acid sequences have acommon structural domain and/or common functional activity. For example,amino acid sequences that comprise a common structural domain that is atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or at least about 100%, identical are definedherein as sufficiently similar. Preferably, variants will besufficiently similar to the amino acid sequence of the preferredpeptides of the present invention, in particular to HTT, variants,derivatives or analogs of either of them. Such variants generally retainthe functional activity of the peptides of the present invention.Variants include peptides that differ in amino acid sequence from thenative and wt peptide, respectively, by way of one or more amino aciddeletion(s), addition(s), and/or substitution(s). These may be naturallyoccurring variants as well as artificially designed ones.

Furthermore, the terms “fragment,” “variant,” “derivative”, and “analog”when referring to antibodies or antibody polypeptides of the presentinvention include any polypeptides which retain at least some of theantigen-binding properties of the corresponding native binding molecule,antibody, or polypeptide. Fragments of polypeptides of the presentinvention include proteolytic fragments, as well as deletion fragments,in addition to specific antibody fragments discussed elsewhere herein.Variants of antibodies and antibody polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of HTT specific binding molecules,e.g., antibodies and antibody polypeptides of the present invention, arepolypeptides which have been altered so as to exhibit additionalfeatures not found on the native polypeptide. Examples include fusionproteins. Variant polypeptides may also be referred to herein as“polypeptide analogs”. As used herein a “derivative” of a bindingmolecule or fragment thereof, an antibody, or an antibody polypeptiderefers to a subject polypeptide having one or more residues chemicallyderivatized by reaction of a functional side group. Also included as“derivatives” are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

Determination of Similarity and/or Identity of Molecules:

“Similarity” between two peptides is determined by comparing the aminoacid sequence of one peptide to the sequence of a second peptide; seeExample 35 as well as FIG. 36. An amino acid of one peptide is similarto the corresponding amino acid of a second peptide if it is identicalor a conservative amino acid substitution. Conservative substitutionsinclude those described in Dayhoff, M. O., ed., The Atlas of ProteinSequence and Structure 5, National Biomedical Research Foundation,Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. Forexample, amino acids belonging to one of the following groups representconservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser,Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His;-Phe, Tyr, Trp, His; and -Asp, Glu.

“Similarity” between two polynucleotides is determined by comparing thenucleic acid sequence of one polynucleotide to the sequence of apolynucleotide. A nucleic acid of one polynucleotide is similar to thecorresponding nucleic acid of a second polynucleotide if it is identicalor, if the nucleic acid is part of a coding sequence, the respectivetriplet comprising the nucleic acid encodes for the same amino acid orfor a conservative amino acid substitution.

The determination of percent identity or similarity between twosequences is preferably accomplished using the mathematical algorithm ofKarlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877. Suchan algorithm is incorporated into the BLASTn and BLASTp programs ofAltschul et al. (1990) J. Mol. Biol. 215: 403-410 available at NCBI(blast.ncbi.nlm.nih.gov/Blast.cgi).

The determination of percent identity or similarity is performed withthe standard parameters of the BLASTn programs for BLAST polynucleotidesearches and BLASTp programs for BLAST protein search, as recommended onthe NCBI webpage and in the “BLAST Program Selection Guide” in respectof sequences of a specific length and composition.

BLAST polynucleotide searches are performed with the BLASTn program.

For the general parameters, the “Max Target Sequences” box may be set to100, the “Short queries” box may be ticked, the “Expect threshold” boxmay be set to 1000 and the “Word Size” box may be set to 7 asrecommended for short sequences (less than 20 bases) on the NCBIwebpage. For longer sequences the “Expect threshold” box may be set to10 and the “Word Size” box may be set to 11. For the scoring parametersthe “Match/mismatch Scores” may be set to 1, −2 and the “Gap Costs” boxmay be set to linear. For the Filters and Masking parameters, the “Lowcomplexity regions” box may not be ticked, the “Species-specificrepeats” box may not be ticked, the “Mask for lookup table only” box maybe ticked, the “DUST Filter Settings” may be ticked and the “Mask lowercase letters” box may not be ticked. In general the “Search for shortnearly exact matches” may be used in this respect, which provides mostof the above indicated settings. Further information in this respect maybe found in the “BLAST Program Selection Guide” published on the NCBIwebpage.

BLAST protein searches are performed with the BLASTp program. For thegeneral parameters, the “Max Target Sequences” box may be set to 100,the “Short queries” box may be ticked, the “Expect threshold” box may beset to 10 and the “Word Size” box may be set to “3”. For the scoringparameters the “Matrix” box may be set to “BLOSUM62”, the “Gap Costs”Box may be set to “Existence: 11 Extension: 1”, the “Compositionaladjustments” box may be set to “Conditional compositional score matrixadjustment”. For the Filters and Masking parameters the “Low complexityregions” box may not be ticked, the “Mask for lookup table only” box maynot be ticked and the “Mask lower case letters” box may not be ticked.

Modifications of both programs, e.g., in respect of the length of thesearched sequences, are performed according to the recommendations inthe “BLAST Program Selection Guide” published in a HTML and a PDFversion on the NCBI webpage.

Polynucleotides:

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding abinding molecule, an antibody, or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operable associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operable associated” or “operablelinked” if induction of promoter function results in the transcriptionof mRNA encoding the desired gene product and if the nature of thelinkage between the two DNA fragments does not interfere with theability of the expression regulatory sequences to direct the expressionof the gene product or interfere with the ability of the DNA template tobe transcribed. Thus, a promoter region would be operable associatedwith a nucleic acid encoding a polypeptide if the promoter was capableof effecting transcription of that nucleic acid. The promoter may be acell-specific promoter that directs substantial transcription of the DNAonly in predetermined cells. Other transcription control elements,besides a promoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operable associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full-length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operable associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

A “binding molecule” as used in the context of the present inventionrelates primarily to antibodies, and fragments thereof, but may alsorefer to other non-antibody molecules that bind to HTT including but notlimited to hormones, receptors, ligands, ankyrins, majorhistocompatibility complex (MHC) molecules, chaperones such as heatshock proteins (HSPs) as well as cell-cell adhesion molecules such asmembers of the cadherin, intergrin, C-type lectin and immunoglobulin(Ig) superfamilies. Thus, for the sake of clarity only and withoutrestricting the scope of the present invention most of the followingembodiments are discussed with respect to antibodies and antibody-likemolecules which represent the preferred binding molecules for thedevelopment of therapeutic and diagnostic agents.

Antibodies:

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is a binding molecule whichcomprises at least the variable domain of a heavy chain, and normallycomprises at least the variable domains of a heavy chain and a lightchain. Basic immunoglobulin structures in vertebrate systems arerelatively well understood; see, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernible to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theTgG class of immunoglobulin molecules. With regard to TgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively. As indicated above, the variable region allows theantibody to selectively recognize and specifically bind epitopes onantigens. That is, the V_(L) domain and V_(H) domain, or subset of thecomplementarity determining regions (CDRs), of an antibody combine toform the variable region that defines a three dimensionalantigen-binding site. This quaternary antibody structure forms theantigen-binding site present at the end of each arm of the Y. Morespecifically, the antigen-binding site is defined by three CDRs on eachof the V_(H) and V_(L) chains. Any antibody or immunoglobulin fragmentwhich contains sufficient structure to specifically bind to HTT isdenoted herein interchangeably as a “binding fragment” or an“immunospecific fragment.”

In naturally occurring antibodies, an antibody comprises sixhypervariable regions, sometimes called “complementarity determiningregions” or “CDRs” present in each antigen-binding domain, which areshort, non-contiguous sequences of amino acids that are specificallypositioned to form the antigen-binding domain as the antibody assumesits three dimensional configuration in an aqueous environment. The“CDRs” are flanked by four relatively conserved “framework” regions or“FRs” which show less inter-molecular variability. The framework regionslargely adopt a β-sheet conformation and the CDRs form loops whichconnect, and in some cases form part of, the β-sheet structure. Thus,framework regions act to form a scaffold that provides for positioningthe CDRs in correct orientation by inter-chain, non-covalentinteractions. The antigen-binding domain formed by the positioned CDRsdefines a surface complementary to the epitope on the immunoreactiveantigen. This complementary surface promotes the non-covalent binding ofthe antibody to its cognate epitope. The amino acids comprising the CDRsand the framework regions, respectively, can be readily identified forany given heavy or light chain variable region by one of ordinary skillin the art, since they have been precisely defined; see, “Sequences ofProteins of Immunological Interest,” Kabat, E., et al., U.S. Departmentof Health and Human Services, (1983); and Chothia and Lesk, J. Mol.Biol., 196 (1987), 901-917, which are incorporated herein by referencein their entireties.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaand Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporatedherein by reference, where the definitions include overlapping orsubsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of anantibody or variants thereof is intended to be within the scope of theterm as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table I as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular hypervariable region orCDR of the human IgG subtype of antibody given the variable region aminoacid sequence of the antibody.

TABLE I CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table I isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the present invention are according tothe Kabat numbering system, which however is theoretical and may notequally apply to every antibody of the present invention. For example,depending on the position of the first CDR the following CDRs might beshifted in either direction.

Unless human-derived monoclonal antibodies or an antigen-bindingfragment, synthetic or biotechnological derivative thereof asparticularly preferred embodiments of the present are referred to,antibodies or antigen-binding fragments, immunospecific fragments,variants, or derivatives thereof of the invention include, but are notlimited to, polyclonal, monoclonal, multispecific, human, humanized,primatized, murinized or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)₂, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a V_(L) or V_(H) domain, fragmentsproduced by a Fab expression library, and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies disclosedherein). ScFv molecules are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

In one embodiment, the antibody of the present invention is not IgM or aderivative thereof with a pentavalent structure. Particular, in specificapplications of the present invention, especially therapeutic use, IgMsare less useful than IgG and other bivalent antibodies or correspondingbinding molecules since IgMs due to their pentavalent structure and lackof affinity maturation often show unspecific cross-reactivities and verylow affinity.

In a particularly preferred embodiment, the antibody of the presentinvention is not a polyclonal antibody, i.e. it substantially consistsof one particular antibody species rather than being a mixture obtainedfrom a plasma immunoglobulin sample.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the invention are HTT binding fragments which comprise anycombination of variable region(s) with a hinge region, CH1, CH2, and CH3domains. Antibodies or immunospecific fragments thereof of the presentinvention may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, donkey, rabbit, goat,guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks).

In one aspect, the antibody of the present invention is a human-derivedmonoclonal antibody isolated from a human, wherein the B cell expressingthe antibody is isolated from a human and in turn the antibody orpreferably the cDNA encoding the variable domain and optionally the cDNAfor the cognate constant domain. Optionally, the framework region of thehuman antibody is aligned and adopted in accordance with the pertinenthuman germ line variable region sequences in the database; see, e.g.,Vbase (vbase.mrc-cpe.cam.ac.uk/) (www.vbase2.org/) hosted by the MRCCentre for Protein Engineering (Cambridge, UK). For example, amino acidsconsidered to potentially deviate from the true germ line sequence couldbe due to the PCR primer sequences incorporated during the cloningprocess. Compared to artificially generated human-like antibodies suchas single chain antibody fragments (scFvs) from a phage displayedantibody library or xenogeneic mice the human monoclonal antibody of thepresent invention is characterized by (i) being obtained using the humanimmune response rather than that of animal surrogates, i.e. the antibodyhas been generated in response to natural HTT in its relevantconformation in the human body, (ii) having protected the individual oris at least significant for the presence of HTT, and (iii) since theantibody is of human origin the risks of cross-reactivity againstself-antigens is minimized. Thus, in accordance with the presentinvention the terms “human monoclonal antibody”, “human monoclonalautoantibody”, “human antibody” and the like are used to denote a HTTbinding molecule which is of human origin, i.e. which has been isolatedfrom a human cell such as a B cell or hybridoma thereof or the cDNA ofwhich has been directly cloned from mRNA of a human cell, for example ahuman memory B cell. A human antibody is still “human”, i.e.human-derived even if amino acid substitutions are made in the antibody,e.g., to improve binding characteristics. In this context, contrary tohumanized antibodies and otherwise human-like antibodies, see also thediscussion infra, the human-derived antibodies of the present inventionare characterized by comprising CDRs which have been seen by human bodyand therefore are substantially devoid of the risk of being immunogenic.Therefore, the antibody of the present invention may still be denotedhuman-derived if at least one, preferably two and most preferably allthree CDRs of one or both the variable light and heavy chain of theantibody are derived from the human antibodies illustrated herein.

In one embodiment the human-derived antibodies of the present inventioncomprises heterologous regions compared to the natural occurringantibodies, e.g. amino acid substitutions in the framework region,constant region exogenously fused to the variable region, differentamino acids at the C- or N-terminal ends and the like.

Antibodies derived from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al. are denoted human-likeantibodies in order distinguish them from truly human antibodies of thepresent invention.

For example, the paring of heavy and light chains of human-likeantibodies such as synthetic and semi-synthetic antibodies typicallyisolated from phage display do not necessarily reflect the originalparing as it occurred in the original human B cell. Accordingly Fab andscFv fragments obtained from recombinant expression libraries ascommonly used in the prior art can be considered as being artificialwith all possible associated effects on immunogenicity and stability.

In contrast, the present invention provides isolated affinity-maturedantibodies from selected human subjects, which are characterized bytheir therapeutic utility and their tolerance in man.

As used herein, the term “rodentized antibody” or “rodentizedimmunoglobulin” refers to an antibody comprising one or more CDRs from ahuman antibody of the present invention; and a human framework regionthat contains amino acid substitutions and/or deletions and/orinsertions that are based on a rodent antibody sequence. When referredto rodents, preferably sequences originating in mice and rats are used,wherein the antibodies comprising such sequences are referred to as“murinized” or “ratinized” respectively. The human immunoglobulinproviding the CDRs is called the “parent” or “acceptor” and the rodentantibody providing the framework changes is called the “donor”. Constantregions need not be present, but if they are, they are usuallysubstantially identical to the rodent antibody constant regions, i.e. atleast about 85% to 90%, preferably about 95% or more identical. Hence,in some embodiments, a full-length murinized human heavy or light chainimmunoglobulin contains a mouse constant region, human CDRs, and asubstantially human framework that has a number of “murinizing” aminoacid substitutions. Typically, a “murinized antibody” is an antibodycomprising a murinized variable light chain and/or a murinized variableheavy chain. For example, a murinized antibody would not encompass atypical chimeric antibody, e.g., because the entire variable region of achimeric antibody is non-mouse. A modified antibody that has been“murinized” by the process of “murinization” binds to the same antigenas the parent antibody that provides the CDRs and is usually lessimmunogenic in mice, as compared to the parent antibody. The aboveexplanations in respect of “murinized” antibodies apply analogously for“rodentized” antibodies, such as “ratinized antibodies”, wherein ratsequences are used instead of the murine.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody or diabody.

As used herein, the term “bispecific” or “bifunctional” antibodymolecule is an antibody molecule that has two different epitope/antigenbinding sites, and accordingly has binding specificities for twodifferent target epitopes. These two epitopes may be epitopes of thesame antigen or of different antigens. In contrast thereto a “bivalentantibody” may have binding sites of identical antigenic specificity.Methods of making a bispecific antibody are known in the art, e.g.chemical conjugation of two different monoclonal antibodies asillustrated in Example 36 or for example, also chemical conjugation oftwo antibody fragments, for example, of two Fab fragments (Brennan etal., Science 229 (1985), 81-83; Nitta et al., Eur. J. Immunol. 19(1989), 1437-1441; Glennie et al., J. Immunol. 139 (1987), 2367-2375;Jung et al., Eur. J. Immunol., 21 (1991), 2431-2435). Alternatively,bispecific antibodies are made recombinantly (Gruber et al., J. Immunol.152 (1994), 5368-5374; Kurucz et al., J. Immunol. 154 (1995), 4576-4582;Mallender and Voss, J. Biol. Chem. 269 (1994), 199-206). Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different binding specificities. Because ofthe random assortment of heavy and light chains, a potential mixture often different antibody structures are produced of which only one has thedesired binding specificity (Milstein and Cuello, Nature 305 (1983),537-540; Lanzavecchia and Scheidegger, Eur. J. Immunol. 17 (1987),105-111. An alternative approach involves fusing the variable domainswith the desired binding specificities to heavy chain constant regionincluding at least part of the hinge region, CH2 and CH3 regions. In oneembodiment the CH1 region containing the site necessary for light chainbinding is present in at least one of the fusions. DNA encoding thesefusions, and if desired the light chains are inserted into separateexpression vectors and are then co-transfected into a suitable hostorganism. It is possible though to insert the coding sequences for twoor all three chains into one expression vector.

In another embodiment, the antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein are composed of asingle polypeptide chain such as scFvs and are to be expressedintracellularly (intrabodies) for potential in vivo therapeutic anddiagnostic applications.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CH1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a V_(L) or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids.

Since a CDR can recognize an antigenic peptide or polypeptide in itstertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, a peptide or polypeptide epitoperecognized by antibodies of the present invention contains a sequence ofat least 4, at least 5, at least 6, at least 7, more preferably at least8, at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 15 to about 30 contiguous or non-contiguous amino acids ofHTT, in particular of a N-terminal, polyP region, P-rich region or theC-terminal region of exon 1.

By “specifically binding”, or “specifically recognizing”, usedinterchangeably herein, it is generally meant that a binding molecule,e.g., an antibody binds to an epitope via its antigen-binding domain,and that the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D”.

Where present, the term “immunological binding characteristics,” orother binding characteristics of an antibody with an antigen, in all ofits grammatical forms, refers to the specificity, affinity,cross-reactivity, and other binding characteristics of an antibody.

By “preferentially binding”, it is meant that the binding molecule,e.g., antibody specifically binds to an epitope more readily than itwould bind to a related, similar, homologous, or analogous epitope.Thus, an antibody which “preferentially binds” to a given epitope wouldmore likely bind to that epitope than to a related epitope, even thoughsuch an antibody may cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it bindssaid first epitope with a dissociation constant (K_(D)) that is lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstantigen preferentially if it binds the first epitope with an affinitythat is at least one order of magnitude less than the antibody's K_(D)for the second epitope. In another non-limiting example, an antibody maybe considered to bind a first epitope preferentially if it binds thefirst epitope with an affinity that is at least two orders of magnitudeless than the antibody's K_(D) for the second epitope.

In another non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it binds thefirst epitope with an off rate (k(off)) that is less than the antibody'sk(off) for the second epitope. In another non-limiting example, anantibody may be considered to bind a first epitope preferentially if itbinds the first epitope with an affinity that is at least one order ofmagnitude less than the antibody's k(off) for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind HTT or afragment, variant or specific conformation thereof with an off rate(k(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹or 10⁻³ sec⁻¹. More preferably, an antibody of the invention may be saidto bind HTT or a fragment, variant or specific conformation thereof withan off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹,5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind HTT or afragment, variant or specific conformation thereof with an on rate(k(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of theinvention may be said to bind HTT or a fragment, variant or specificconformation thereof with an on rate (k(on)) greater than or equal to10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷M⁻¹ sec⁻¹.

A binding molecule, e.g., an antibody is said to competitively inhibitbinding of a reference antibody to a given epitope if it preferentiallybinds to that epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitionmay be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of a bindingmolecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. (1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen; see, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valences of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity. The affinity or avidity of an antibody foran antigen can be determined experimentally using any suitable method;see, for example, Berzofsky et al., “Antibody-Antigen Interactions” InFundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y(1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y(1992), and methods described herein. General techniques for measuringthe affinity of an antibody for an antigen include ELISA, RIA, andsurface plasmon resonance. The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions, e.g., salt concentration, pH. Thus, measurements of affinityand other antigen-binding parameters, e.g., K_(D), IC₅₀, are preferablymade with standardized solutions of antibody and antigen, and astandardized buffer.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their cross-reactivity. As used herein, theterm “cross-reactivity” refers to the ability of an antibody, specificfor one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to HTT and/or mutated,misfolded, and/or aggregated HTT species and/or fragments thereof.

Preferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M,5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M,5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the V_(H) domain and is amino terminal to the hinge regionof an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit). The CH2 domain is unique in that it isnot closely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains; see Roux et al., J.Immunol. 161 (1998), 4083-4090.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the terms “linked”, “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of mRNA into polypeptide(s).If the final desired product is a biochemical, expression includes thecreation of that biochemical and any precursors. Expression of a geneproduces a “gene product.” As used herein, a gene product can be eithera nucleic acid, e.g., a messenger RNA produced by transcription of agene, or a polypeptide which is translated from a transcript. Geneproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation, or polypeptideswith post translational modifications, e.g., methylation, glycosylation,the addition of lipids, association with other protein subunits,proteolytic cleavage, and the like.

As used herein, the term “sample” refers to any biological materialobtained from a subject or patient. In one aspect, a sample can compriseblood, peritoneal fluid, CSF, saliva or urine. In other aspects, asample can comprise whole blood, blood plasma, blood serum, B cellsenriched from blood samples, and cultured cells (e.g., B cells from asubject). A sample can also include a biopsy or tissue sample includingneural tissue. In still other aspects, a sample can comprise whole cellsand/or a lysate of the cells. Blood samples can be collected by methodsknown in the art. In one aspect, the pellet can be resuspended byvortexing at 4° C. in 200 μl buffer (20 mM Tris, pH. 7.5, 0.5% Nonidet,1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, IX Sigma Protease Inhibitor, and IXSigma Phosphatase Inhibitors 1 and 2). The suspension can be kept on icefor 20 min. with intermittent vortexing. After spinning at 15,000×g for5 min at about 4° C., aliquots of supernatant can be stored at about−70° C.

Diseases:

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein and comprise any undesired physiological changein a subject, an animal, an isolated organ, tissue or cell/cell culture.

Huntington's disease (HD) is an autosomal dominant, progressiveneurodegenerative disorder characterized by the expansion of a CAGtrinucleotide repeat within the huntingtin (HTT) gene (Huntington'sDisease Collaborative Research Group, Cell 72(6) (1993), 971-983),wherein the pathogenic threshold of this expansion is approximately 37repeats, whereas fewer repeats below this number do not result inpathogenesis, see e.g. Trottier et al., Nature 378(6555) (1995),403-406. The expansion of the CAG trinucleotide repeat results in anexpanded polyglutamine (Poly-Gln, Poly-Q) tract in the amino terminus ofthe huntingtin protein (HTT), which is associated with the aggregationof HTT. However, the precise mechanism leading to the accumulation ofHTT and its associated symptoms has not been elucidated so far.

Studies have shown that both flanking regions of the polyglutamine(Poly-Q) tract, i.e. amino-terminal region consisting of an amphipathicalpha-helical targeting domain and the carboxy-terminal regioncharacterized by two proline tracts (Poly-P region) and aleucine-proline-rich tract (P-rich region) seem to be critical inmediating the toxicity of the mutated HTT, see e.g. Caron et al., PNAS110 (2013), 14610-14615.

The mechanism contributing to the pathological symptoms of HD, such ashyperkinesia, hypokinesia, mental and movement disorders includingdisturbances of affect and the drive, lack of motor persistence,thoughtless and impulsive behavior, resignation and depression,disorders of visual information processing, subcortical dementia, lossof cognitive abilities, disorientation and paucity of speech, delusions,restlessness of the arms, legs, face, head and the trunk, choreichyperkinesis, dysarthria, dysphagia, anarthria, dystonias, has not beenelucidated so far. Possible mechanisms include but are not limited to areduced flexibility of the hinge region due to the expanded Poly-Q tractof HTT as well as to proteases which lead to the formation of differentHTT fragments due to the expanded Poly-Q tract.

Since the antibodies of the present invention have been shown to betherapeutically effective in a HD mouse model, see e.g. Example 24 andFIG. 17 as well as Example 34 and FIG. 34 and in addition are capable ofbinding to HTT amyloids in tissue sections from HD patients, see e.g.Example 31 and FIG. 27-30, the human-derived antibodies andbiotechnological derivatives thereof are useful in both the treatmentand diagnosis of HD and the above-mentioned symptoms. Therefore, in oneembodiment of the present invention the antibodies of the presentinvention, binding molecules having substantially the same bindingspecificities of any one thereof, the polynucleotides, the vectors orthe cells of the present invention are used for preparation of apharmaceutical or diagnostic composition for prophylactic and/ortherapeutic treatment of HD, in particular HTT amyloidosis diseasesand/or disorders, for monitoring disease progression and/or treatmentresponse, and for the diagnosis of diseases associated with HTTamyloidosis.

Treatment:

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development of cardiacdeficiency. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which themanifestation of the condition or disorder is to be prevented.

If not stated otherwise the term “drug,” “medicine,” or “medicament” areused interchangeably herein and shall include but are not limited to all(A) articles, medicines and preparations for internal or external use,and any substance or mixture of substances intended to be used fordiagnosis, cure, mitigation, treatment, or prevention of disease ofeither man or other animals; and (B) articles, medicines andpreparations (other than food) intended to affect the structure or anyfunction of the body of man or other animals; and (C) articles intendedfor use as a component of any article specified in clause (A) and (B).The term “drug,” “medicine,” or “medicament” shall include the completeformula of the preparation intended for use in either man or otheranimals containing one or more “agents,” “compounds”, “substances” or“(chemical) compositions” as and in some other context also otherpharmaceutically inactive excipients as fillers, disintegrants,lubricants, glidants, binders or ensuring easy transport,disintegration, disaggregation, dissolution and biological availabilityof the “drug,” “medicine,” or “medicament” at an intended targetlocation within the body of man or other animals, e.g., at the skin, inthe stomach or the intestine. The terms “agent,” “compound”, or“substance” are used interchangeably herein and shall include, in a moreparticular context, but are not limited to all pharmacologically activeagents, i.e. agents that induce a desired biological or pharmacologicaleffect or are investigated or tested for the capability of inducing sucha possible pharmacological effect by the methods of the presentinvention.

By “subject” or “individual” or “animal” or “patient” or “mammal”, ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

Pharmaceutical Carriers:

Pharmaceutically acceptable carriers and administration routes can betaken from corresponding literature known to the person skilled in theart. The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols 2ndEdition by Robinson et al., Humana Press, Totowa, N.J., USA, 2003;Banga, Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems. 2nd Edition by Taylor and Francis. (2006), ISBN:0-8493-1630-8. Examples of suitable pharmaceutical carriers are wellknown in the art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by well-known conventional methods. These pharmaceuticalcompositions can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways. Examples include administering a composition containing apharmaceutically acceptable carrier via oral, intranasal, rectal,topical, intraperitoneal, intravenous, intramuscular, subcutaneous,subdermal, transdermal, intrathecal, and intracranial methods. Aerosolformulations such as nasal spray formulations include purified aqueousor other solutions of the active agent with preservative agents andisotonic agents. Such formulations are preferably adjusted to a pH andisotonic state compatible with the nasal mucous membranes.Pharmaceutical compositions for oral administration, such as singledomain antibody molecules (e.g., “Nanobodies™”) etc. are also envisagedin the present invention. Such oral formulations may be in tablet,capsule, powder, liquid or semi-solid form. A tablet may comprise asolid carrier, such as gelatin or an adjuvant. Formulations for rectalor vaginal administration may be presented as a suppository with asuitable carrier; see also O'Hagan et al., Nature Reviews, DrugDiscovery 2(9) (2003), 727-735. Further guidance regarding formulationsthat are suitable for various types of administration can be found inRemington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985) and corresponding updates. For abrief review of methods for drug delivery see Langer, Science 249(1990), 1527-1533.

II. Antibodies of the Present Invention

The present invention generally relates to human-derived anti-HTTantibodies and HTT-binding fragments thereof, which preferablydemonstrate the immunological binding characteristics and/or biologicalproperties as outlined for the antibodies illustrated in the Examples.In accordance with the present invention human monoclonal antibodiesspecific for HTT were cloned from B cells of a pool of healthy humansubjects. However, in another embodiment of the present invention, thehuman monoclonal anti-HTT antibodies might also be cloned from B cellsof patients showing symptoms of a disease and/or disorder associatedwith HTT amyloidosis.

In the course of the experiments performed in accordance with thepresent invention, antibodies present in the conditioned media ofcultured human memory B cell were evaluated for their capacity to bindto HTT and to more than 10 other proteins including bovine serum albumin(BSA); see Examples 8, 13, 18, 31 and 33. Only the B-cell supernatantsable to bind to the HTT protein but not to any of the other proteins inthe screen were selected for further analysis, including determinationof the antibody class and light chain subclass. The selected B-cellswere then processed for antibody cloning.

In brief, this consisted in the extraction of messenger RNAs from theselected B-cells, retro-transcription by RT-PCR, amplification of theantibody-coding regions by PCR, cloning into plasmid vectors andsequencing. Selected human antibodies were then produced by recombinantexpression in HEK293 or CHO cells and purification, and subsequentlycharacterized for their capacity to bind human HTT protein. Thecombination of various tests, e.g. recombinant expression of theantibodies in HEK293 or CHO cells and the subsequent characterization oftheir binding specificities towards human HTT protein, and theirdistinctive binding to pathologically mutated and/or aggregated formsthereof confirmed that for the first time human antibodies have beencloned that are highly specific for HTT and distinctively recognize andselectively bind the pathologically aggregated forms of HTT protein. Insome cases, mouse chimeric antibodies were also generated on the basisof the variable domains of the human antibodies of the presentinvention.

Thus, the present invention generally relates to recombinanthuman-derived monoclonal anti-HTT antibodies and HTT-binding fragments,synthetic and biotechnological derivatives and variants thereof. In oneembodiment of the invention, the antibody is capable of binding humanHTT.

In one embodiment of the present invention the antibody specificallybinds an epitope in a polyP-region of HTT, which comprises the aminoacid sequence PPPPPPPP (NI-302.33C11; NI-302.44D7; NI-302.7A8;NI-302.3D8; NI-302.46C9) (SEQ ID Nos. 139, 151, 154, 158, 161), aminoacid sequence PPPPPP (NI-302.11H6, NI-302.18A1, NI-302.52C9 (SEQ IDNos.: 157, 159, 160), amino acid sequence PPPPPPPPPPP (NI-302.74C11,NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.37C12,NI-302.55D8, NI-302.78H12, NI-302.71F6 (SEQ ID Nos.: 146, 147, 148, 149,150, 152, 153, 155, 156), an epitope in the P-rich-region whichcomprises the amino acid sequence PQPPPQAQPL (NI-302.63F3 SEQ ID No.140, NI-302.64E5 SEQ ID No. 200), the amino acid sequence PPPQLPQPPP(NI-302.31F11, SEQ ID No. 141), the amino acid sequence QAQPLLPQPQPPPPP(NI-302.2A2; SEQ ID No. 142), or the amino acid sequence PPPQLPQPPPQAQPL(NI302.15D3; SEQ ID No. 143), an epitope in the C-terminal region whichcomprise the amino acid sequence PPGPAVAEEPLHRP (NI-302.35C1, SEQ ID No.145) or PPPGPAVAEEPLH (NI-302.72F10, SEQ ID No. 202), an epitope in theN-terminal region which comprises the amino acid sequence KAFESLKSFQ(NI-NI-302.15E8, SEQ ID No. 144) or an epitope in the P/Q-rich-regionwhich comprises the amino acid sequence QQQQQQQQQPPP (NI-302.7D8 SEQ IDNo. 201), or a conformational epitope.

In another embodiment, the present invention is directed to an anti-HTTantibody, or antigen-binding fragment, variant or biotechnologicalderivatives thereof, where the antibody specifically binds to the sameepitope in a polyP-region of HTT as a reference antibody selected fromthe group consisting of NI-302.33C11, NI-302.74C11, NI-302.15F9,NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12,NI-302.55D8, NI-302.7A8, 302.78H12, NI-302.71F6, NI-302.11H6,NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9. Epitopemapping identified a sequence within the polyP-region of human HTTincluding amino acids PPPPPPPPPPP (SEQ ID Nos.: 146, 147, 148, 149, 150,152, 153, 155, 156) as the unique linear epitope recognized byantibodies NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4,NI-302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI-302.71F6 ofthis invention. Additionally, epitope mapping identified a sequencewithin the polyP-region of human HTT including amino acids PPPPPPPP (SEQID Nos. 139, 151, 154, 158, 161) as the unique linear epitope recognizedby antibodies NI-302.33C11, NI-302.44D7, NI-302.7A8, NI-302.3D8,NI-302.46C9 of this invention, and amino acids PPPPPP (SEQ ID Nos.: 157,159, 160) as the unique linear epitope recognized by antibodiesNI-302.11H6, NI-302.18A1, NI-302.52C9 of this invention. Therefore, inone embodiment the antibody of the present invention is provided,wherein the antibody specifically binds to an epitope in a polyP-regionof HTT, which comprises the amino acid sequence PPPPPPPPPPP (SEQ IDNos.: 146, 147, 148, 149, 150, 152, 153, 155, 156), PPPPPPPP (SEQ IDNos. 139, 151, 154, 158, 161), or PPPPPP (SEQ ID Nos.: 157, 159, 160).

In one embodiment, the present invention is directed to an anti-HTTantibody, or antigen-binding fragment, variant or biotechnologicalderivatives thereof, where the antibody specifically binds to the sameepitope in the P-rich region of HTT as a reference antibody selectedfrom the group consisting of NI-302.63F3, NI-302.31F11, NI-302.2A2, andNI-302.15D3. Epitope mapping identified a sequence within theP-rich-region of human HTT including amino acids PQPPPQAQPL (SEQ ID No.140) as the unique linear epitope recognized by antibody NI-302.63F3 ofthis invention, PPPQLPQPPP (SEQ ID No. 141), as the unique linearepitope recognized by antibody NI-302.31F11 of this invention,PPPQLPQPPP (SEQ ID No. 141), as the unique linear epitope recognized byantibody NI-302.31F11 of this invention, QAQPLLPQPQPPPPP (SEQ ID No.142) as the unique linear epitope recognized by antibody NI-302.2A2,PPPQLPQPPPQAQPL (SEQ ID No. 143) as the unique linear epitope recognizedby antibody NI302.15D3, PQPPPQAQPL as the unique linear epitoperecognized by antibody NI302.64E5. Therefore, in one embodiment theantibody of the present invention is provided, wherein the antibodyspecifically binds to an epitope in the P-rich-region of HTT whichcomprises the amino acid sequence PQPPPQAQPL (SEQ ID No. 140),PPPQLPQPPP (SEQ ID No. 141), QAQPLLPQPQPPPPP (SEQ ID No. 142),PPPQLPQPPPQAQPL (SEQ ID No. 143).

In another embodiment the present invention is directed to an anti-HTTantibody, or antigen-binding fragment, variant or biotechnologicalderivatives thereof, wherein the antibody specifically binds to the sameepitope in the polyQ/polyP-region of HTT as reference antibodyNI-302.7D8. Epitope mapping identified a sequence within theQ/P-rich-region of human HTT including amino acids QQQQQQQPPP (SEQ IDNo. 201) as the unique linear epitope recognized by antibody NI-302.7D8of this invention. Therefore, in one embodiment the antibody of thepresent invention is provided, wherein the antibody specifically bindsto an epitope in in the polyQ/polyP-region of HTT which comprises theamino acid sequence QQQQQQQPPP (SEQ ID No. 201)

In one embodiment, the present invention is directed to an anti-HTTantibody, or antigen-binding fragment, variant or biotechnologicalderivatives thereof, where the antibody specifically binds to the sameepitope in the C-terminal region of HTT as a reference antibody selectedfrom the group consisting of NI-302.35C1. Epitope mapping identified asequence within the C-terminal region of human HTT including amino acidsPPGPAVAEEPLHRP (SEQ ID No. 145) as the unique linear epitope recognizedby antibody NI-302.35C1 of this invention. Therefore, in one embodimentthe antibody of the present invention is provided, wherein the antibodyspecifically binds to an epitope in in the C-terminal region of HTTwhich comprises the amino acid sequence PPGPAVAEEPLHRP (SEQ ID No. 145).

In a further embodiment, the present invention is directed to ananti-HTT antibody, or antigen-binding fragment, variant orbiotechnological derivatives thereof, where the antibody specificallybinds to the same epitope in the C-terminal region of HTT as referenceantibody NI-302.72F10. Epitope mapping identified a sequence within theC-terminal region of human HTT including amino acids PPPGPAVAEEPLH (SEQID No. 202) as the unique linear epitope recognized by antibodyNI-302.72F10 of this invention. Therefore, in one embodiment theantibody of the present invention is provided, wherein the antibodyspecifically binds to an epitope in in the C-terminal region of HTTwhich comprises the amino acid sequence PPPGPAVAEEPLH (SEQ ID No. 202).

In another embodiment, the present invention is directed to an anti-HTTantibody, or antigen-binding fragment, variant or biotechnologicalderivatives thereof, where the antibody specifically binds to the sameepitope in the N-terminal region of HTT as reference antibodyNI-302.15E8. Epitope mapping identified a sequence within the N-terminalregion of human HTT including amino acids KAFESLKSFQ (SEQ ID No. 144) asthe unique linear epitope recognized by antibody NI-302.15E8 of thisinvention. Therefore, in one embodiment the antibody of the presentinvention is provided, wherein the antibody specifically binds to anepitope in in the N-terminal region of HTT which comprises the aminoacid sequence KAFESLKSFQ (SEQ ID No. 144).

In one embodiment, the present invention is directed to an anti-HTTantibody, or antigen-binding fragment, variant or biotechnologicalderivatives thereof, where the antibody specifically binds to the sameepitope of HTT exon 1 as a reference antibody selected from the groupconsisting of NI-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1 whichhave been shown not to bind to linear peptides of HTT exon 1 butaggregated HTT exon 1 proteins with 21 or 49 polyQ (HD21 and HD49) withhigh affinity and an EC₅₀ value in the subnanomolar range; see, e.g.,Example 25 and FIG. 20 for overview. Therefore, in one preferredembodiment the antibody of the present invention specifically bindsaggregated forms of HTT, in particular protein aggregates derived fromHTT exon 1 with an EC₅₀ value of below 1 nM, preferably below 0.1 nM andmost preferably below 0.01 nM.

Furthermore, without intending to be bound by initial experimentalobservations as demonstrated in the Examples and shown in Figures, thehuman monoclonal NI-302.33C11, NI-302.63F3, NI-302.35C1, NI-302.31F11,NI-302.6N9, NI-302.46C9, NI-302.8F1, NI-302.74C11, NI-302.15F9,NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.55D8,NI-302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, andNI.302-64E5 and NI.302-72F10 anti-HTT antibodies of the presentinvention are preferably characterized in specifically binding topathological mutated and/or aggregated HTT and to substantially smalleraffinity recognizing HTT in the physiological form, see e.g. Examples 7,13, 18 and FIGS. 3, 7, 11, 21, 32. Hence, the present invention providesa set of human anti-HTT antibodies with binding properties particularlyuseful for diagnostic and therapeutic purposes.

Thus, in one embodiment the present invention provides antibodies whichare capable of specifically binding pathologically aggregated forms ofHTT. However, in addition or alternatively, the antibodies of thepresent invention which are capable to bind to a polyP-region or aP-rich region of HTT exon 1 may be also utilized in other applications.In particular, these antibodies are not limited to HTT but can also bindto other targets showing also a polyP-tract or a P-rich region.

In one embodiment, the antibody of the present invention exhibits thebinding properties of the exemplary NI-302.33C11, NI-302.63F3,NI-302.35C1, NI.302-7D8 and NI.302-72F10 antibodies as described in theExamples. The anti-HTT antibody of the present invention preferentiallyrecognizes pathologically altered HTT, such as mutated and/or aggregatedHTT species and fragments thereof rather than physiological HTT. Thus,in one embodiment, the antibody of the present invention does notsubstantially recognize physiological HTT species.

The term “does not substantially recognize” when used in the presentapplication to describe the binding affinity of a molecule of a groupcomprising an antibody, a fragment thereof or a binding molecule for aspecific target molecule, antigen and/or conformation of the targetmolecule and/or antigen means that the molecule of the aforementionedgroup binds said molecule, antigen and/or conformation with a bindingaffinity which is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold or 9-fold less than the binding affinity of the moleculeof the aforementioned group for binding another molecule, antigen and/orconformation. Very often the dissociation constant (KD) is used as ameasure of the binding affinity. Sometimes, it is the EC₅₀ on a specificassay as for example an ELISA assay that is used as a measure of thebinding affinity. Preferably the term “does not substantially recognize”when used in the present application means that the molecule of theaforementioned group binds said molecule, antigen and/or conformationwith a binding affinity which is at least or 10-fold, 20-fold, 50-fold,100-fold, 1000-fold or 10000-fold less than the binding affinity of saidmolecule of the aforementioned group for binding to another molecule,antigen and/or conformation.

As described above, the aggregation of HTT in HD is suggested to occurdue to an extension of the poly-glutamine tract within the HTT exon 1.In particular, it has been shown that HD mainly occurs in patientshaving a threshold over the 35-40 glutamine residues in length in theHTT. Accordingly, as shown in Example 3, aggregated and solubleconstruct of HTT exon 1 with 21, 35 or 49 polyQ repeats were generatedin order to identify the utility of the anti-HTT-antibodies of thepresent invention to specifically bind to pathological altered HTT.

The term HDX as used in the following describes the HTT constructs whichwere generated in accordance with Example 3. Particularly the X denotesthe number of glutamine repeats (Qs), e.g. HTT exon 1 protein with 21polyQ repeats will be denoted HD21.

Utilizing the constructs as described in the Examples, it could be shownthat the anti-HTT antibody of the present invention in addition, oralternatively, binds to pathologically, disease causing and/or mutatedand/or aggregated forms of human HTT. In this context, the bindingaffinities may be in the range as shown for the exemplary NI-302.33C11,NI-302.63F3, NI-302.35C1, NI-302.31F111, NI-302.6N9, NI-302.46C9,NI-302.8F1, NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4,NI-302.22H9, NI-302.44D7, NI-302.55D8, NI-302.7A8, NI-302.78H12,NI-302.71F6, NI-302.11H6, and NI-302.3D8 antibodies in FIG. 3(A), 7(A),11(A), 14(A), respective FIGS. 19, 20 and 31, i.e. having half maximaleffective concentrations (EC₅₀) of about 1 pM to 250 nM, preferably anEC₅₀ of about 25 pM to 50 nM, most preferably an EC₅₀ of about 0.05 nMto 30 nM for human aggregated HD49-HTT and aggregated recombinantHD49-HTT, or an EC₅₀ of about 0.05 nM to 5 nM for human aggregatedHD21-HTT and aggregated recombinant HD21-HTT.

In particular, the anti-HTT antibody, binding fragment orbiotechnological derivative thereof has a binding affinity correspondingto an EC₅₀ value of ≤20 nM, preferably ≤10 nM and most preferably ≤1 nMfor binding aggregated HD49 HTT and/or of ≤40 nM, preferably ≤10 nM andmost preferably 1 nM for binding HD21 HTT; see FIGS. 3, 7, 11, 19 and31.

HTT aggregation associated with the development of HD is most frequentlyassociated with poly-glutamine (polyQ) tracts of >35 repeats. As shownin the present invention, the anti-HTT antibodies described hereinshowed high binding efficiency to HD tracts with higher repetitions, seee.g. Examples 7, 13, 18, 31 and 33. Therefore, in one embodiment of thepresent invention the anti-HTT antibody, HTT-binding molecule, fragment,synthetic or biotechnological variant thereof binds to HTT with expandedpoly-glutamine (Q) tract. In a preferred embodiment it binds to HTT withmore than 35 repeats. In a particular preferred embodiment of thepresent invention, the antibody binds to HTT with expandedpoly-glutamine (Q) tract consisting of 49 (HD49) repeats over 35 repeats(HD35) and more over 21 repeats (HD21).

However, in accordance with the present invention also anti-HTTantibodies, HTT-binding molecules, fragments, synthetic orbiotechnological variants thereof binding to poly-glutamine (polyQ)tracts under 35 (HD35) are described. Therefore, in one embodiment ofthe present invention, the antibody, binding molecule or variantsthereof binds to HTT showing “normal” polyQ tracts. In particular, theantibody is capable of binding to HTT with polyQ tracts<35 repeats(HD35).

Some antibodies are able to bind to a wide array of biomolecules, e.g.,proteins. As the skilled artisan will appreciate, the term specific isused herein to indicate that other biomolecules than HTT proteins orfragments thereof do not significantly bind to the antigen-bindingmolecule, e.g., one of the antibodies of the present invention.Preferably, the level of binding to a biomolecule other than HTT resultsin a binding affinity which is at most only 20% or less, 10% or less,only 5% or less, only 2% or less or only 1% or less (i.e. at least 5,10, 20, 50 or 100 fold lower, or anything beyond that) of the affinityto HTT, respectively; see e.g., FIG. 20.

In one embodiment, the anti-HTT antibody of the present invention bindspreferentially to aggregated forms of HTT and/or fragments, derivatives,fibrils and/or oligomers thereof. In another embodiment the anti-HTTantibody of the present invention preferentially binds to both nativeHTT and pathologically mutated and/or aggregated forms of HTT.

In a further embodiment of the present invention, the anti-HTT antibodyor HTT-binding fragment, synthetic or biotechnological derivativethereof is a bispecific antibody. Thus, the antibody of the presentinvention may be capable of recognizing at least two distinct epitopeseither on the same or on different antigens; see also, supra.

In one embodiment, at least one binding site/domain of the bispecificantibody specifically recognizes an epitope in a polyP-region of HTT,which comprises the amino acid sequence PPPPPPPP (NI-302.33C11;NI-302.44D7; NI-302.7A8; NI-302.3D8; NI-302.46C9) (SEQ ID Nos. 139, 151,154, 158, 161), amino acid sequence PPPPPP (NI-302.11H6, NI-302.18A1,NI-302.52C9 (SEQ ID Nos.: 157, 159, 160), amino acid sequencePPPPPPPPPPP (NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4,NI-302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI-302.71F6 (SEQID Nos.: 146, 147, 148, 149, 150, 152, 153, 155, 156), an epitope in theP-rich-region which comprises the amino acid sequence PQPPPQAQPL(NI-302.63F3 SEQ ID No. 140, NI-302.64E5 SEQ ID No. 200), the amino acidsequence PPPQLPQPPP (NI-302.31F11, SEQ ID No. 141), the amino acidsequence QAQPLLPQPQPPPPP (NI-302.2A2; SEQ ID No. 142), or the amino acidsequence PPPQLPQPPPQAQPL (NI302.15D3; SEQ ID No. 143), an epitope in theC-terminal region which comprise the amino acid sequence PPGPAVAEEPLHRP(NI-302.35C1, SEQ ID No. 145) or PPPGPAVAEEPLH (NI-302.72F10, SEQ ID No.202), an epitope in the N-terminal region which comprises the amino acidsequence KAFESLKSFQ (NI-302.15E8, SEQ ID No. 144), an epitope in theP/Q-rich-region which comprises the amino acid sequence QQQQQQQQQPPP(NI-302.7D8 SEQ ID No. 201), or a conformational epitope recognized byany one of antibodies NI-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1.

As mentioned before, accumulation of polyglutamine (poly-Gln,polyQ)-containing HTT protein aggregates in neuronal intranuclearinclusions is a hallmark of the progressive neurodegenerative disorderHuntington's disease (HD). Electron micrographs of these aggregatesrevealed fibrillar structures showing a closely related morphology as inB-amyloid fibrils in Alzheimer's disease, see e.g. Caughey et al.,Trends Cell Biol. 7 (1997), 56-62 and Caputo et al., Arch. Biochem.Biophys. 292 (1992), 199-205, suggesting that HD, wherein degenerativeprocess primarily involves medium spiny striatal neurons and corticalneurons leading to dysfunction and subsequently neuronal loss, tissuedamage due to excitotoxicity, mitochondrial damage, free radicals, andpossibly also inflammatory mechanisms including microglia activation andfurther progressive nature of symptoms, are a result of toxic amyloidfibrillogenesis. Therefore, in one embodiment the antibody of thepresent invention is useful for the treatment of Huntingtion's disease(HD) and symptoms thereof.

So far, intracellularly expressed antibodies (intrabodies) have beendescribed and considered as therapeutic tools in HD which perturb theHTT function, see e.g. Ali et al. in Neurobiology of Huntington'sDisease: Applications to Drug Discovery, Lo et al., Chapter 10, CRCPress (2011). Although these intrabodies showed a positive effect on theaggregation and cell death induced by HTT in cell based assays, see e.g.Khoshnan et al., Proc Natl Acad Sci USA. 99 (2002), 1002-1007, onedisadvantage in their therapeutic utility is the route ofadministration. In particular, the preferred method for the delivery ofthe therapeutic intrabodies to the brain is a viral vector-based genetherapy. However, a major disadvantage of using this kind ofadministration is among other the high host immunogenicity. Therefore,non-viral methods utilizing other routes of administration as arepreferably used in the therapeutic or diagnostic approaches. Theantibodies of the present invention have been shown to attenuate thedendritic spine density loss upon addition to the culture medium, i.e.extracellularly. Therefore, in contrast to the intrabodies describedbefore, the antibodies of the present invention can be expected to beefficacious following therapeutically preferred administration routes.Accordingly, in one embodiment of the present invention the antibody isadministrated by a subcutaneous injection (s.c.), intravenous injection(i.v.), intramuscular injection (i.m.), intraperitoneal (i.p.),intrathecal, jet injection, wherein the radius of action is not limitedto the intracellular expression of the antibody.

As already mentioned before, and as shown in Example 24 and FIG. 17 thetherapeutic utility of the antibodies of the present invention has beenshown. In particular, it has been shown that the anti-HTT antibodies ofthe present invention are capable of attenuating the dendritic spinedensity loss. Therefore, in one embodiment of the present invention theant-HTT antibody, the HTT-binding fragment, synthetic orbiotechnological derivative thereof leads to an attenuation of spinedensity loss.

Furthermore, the therapeutic utility of the antibodies of the presentinvention has been demonstrated in Example 34 and FIG. 34. Inparticular, it has been shown that the anti-HTT antibodies of thepresent invention improve behavioral recovery during task-specifictraining and enhance loco-motor ability. Therefore, in one embodiment ofthe present invention the ant-HTT antibody, the HTT-binding fragment,synthetic or biotechnological derivative thereof leads to an improvementof behavioral performance during task-specific training and enhancementof sensorimotor ability.

The present invention is also drawn to an antibody, or antigen-bindingfragment, variant or derivatives thereof, where the antibody comprisesan antigen-binding domain identical to that of an antibody selected fromthe group consisting of NI-302.33C11, NI-302.63F3, NI-302.35C1,NI-302.31F11, NI-302.2A2, NI-302.6N9, NI-302.74C11, NI-302.15F9,NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12,NI-302.55D8, NI-302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6,NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9,NI-302.15E8, NI-302.15D3, NI-302.64E5, NI-302.7D8, NI-302.72F10,NI-302.12H2, NI-302.8M1 and NI-3024A6.

The present invention further exemplifies several binding molecules,e.g., antibodies and binding fragments thereof, recognizing apolyP-region of HTT, which may be characterized by comprising in theirvariable region, e.g., binding domain at least one complementaritydetermining region (CDR) of the V_(H) and/or V_(L) variable regioncomprising any one of the amino acid sequences depicted in FIG. 1. Thecorresponding nucleotide sequences encoding the above-identifiedvariable regions are set forth in Table II below. Exemplary sets of CDRsof the above amino acid sequences of the V_(H) and/or V_(L) region aredepicted in FIG. 1. However, as discussed in the following the personskilled in the art is well aware of the fact that in addition oralternatively CDRs may be used, which differ in their amino acidsequence from those set forth in FIG. 1 by one, two, three or even moreamino acids in case of CDR2 and CDR3. Therefore, in one embodiment theantibody of the present invention or a HTT-binding fragment thereof isprovided comprising in its variable region at least one complementaritydetermining region (CDR) as depicted in FIG. 1 and/or one or more CDRsthereof comprising one or more amino acid substitutions.

Further the present invention exemplifies several binding molecules,e.g., antibodies and binding fragments thereof, recognizing the P-richregion of HTT which may be characterized by comprising in their variableregion, e.g., binding domain at least one complementarity determiningregion (CDR) of the V_(H) and/or V_(L) variable region comprising anyone of the amino acid sequences depicted in FIG. 1. The correspondingnucleotide sequences encoding the above-identified variable regions areset forth in Table III below. Exemplary sets of CDRs of the above aminoacid sequences of the V_(H) and/or V_(L) region are depicted in FIG. 1.However, as discussed in the following the person skilled in the art iswell aware of the fact that in addition or alternatively CDRs may beused, which differ in their amino acid sequence from those set forth inFIG. 1 by one, two, three or even more amino acids in case of CDR2 andCDR3. Therefore, in one embodiment the antibody of the present inventionor a HTT-binding fragment thereof is provided comprising in its variableregion at least one complementarity determining region (CDR) as depictedin FIG. 1 and/or one or more CDRs thereof comprising one or more aminoacid substitutions.

The present invention in addition exemplifies several binding molecules,e.g., antibodies and binding fragments thereof, recognizing theC-terminal region of HTT which may be characterized by comprising intheir variable region, e.g., binding domain at least one complementaritydetermining region (CDR) of the V_(H) and/or V_(L) variable regioncomprising any one of the amino acid sequences depicted in FIG. 1. Thecorresponding nucleotide sequences encoding the above-identifiedvariable regions are set forth in Table IV below. Exemplary sets of CDRsof the above amino acid sequences of the V_(H) and/or V_(L) region aredepicted in FIG. 1. However, as discussed in the following the personskilled in the art is well aware of the fact that in addition oralternatively CDRs may be used, which differ in their amino acidsequence from those set forth in FIG. 1 by one, two, three or even moreamino acids in case of CDR2 and CDR3. Therefore, in one embodiment theantibody of the present invention or a HTT-binding fragment thereof isprovided comprising in its variable region at least one complementaritydetermining region (CDR) as depicted in FIG. 1 and/or one or more CDRsthereof comprising one or more amino acid substitutions.

Additionally, the present invention exemplifies several bindingmolecules, e.g., antibodies and binding fragments thereof, recognizingthe N-terminal-region of HTT which may be characterized by comprising intheir variable region, e.g., binding domain at least one complementaritydetermining region (CDR) of the V_(H) and/or V_(L) variable regioncomprising any one of the amino acid sequences depicted in FIG. 1. Thecorresponding nucleotide sequences encoding the above-identifiedvariable regions are set forth in Table VI below. Exemplary sets of CDRsof the above amino acid sequences of the V_(H) and/or V_(L) region aredepicted in FIG. 1. However, as discussed in the following the personskilled in the art is well aware of the fact that in addition oralternatively CDRs may be used, which differ in their amino acidsequence from those set forth in FIG. 1 by one, two, three or even moreamino acids in case of CDR2 and CDR3. Therefore, in one embodiment theantibody of the present invention or a HTT-binding fragment thereof isprovided comprising in its variable region at least one complementaritydetermining region (CDR) as depicted in FIG. 1 and/or one or more CDRsthereof comprising one or more amino acid substitutions.

In one embodiment, the antibody of the present invention is any one ofthe antibodies comprising an amino acid sequence of the V_(H) and/orV_(L) region as depicted in FIG. 1 or a V_(H) and/or V_(L) regionthereof comprising one or more amino acid substitutions. Preferably, theantibody of the present invention is characterized by the preservationof the cognate pairing of the heavy and light chain as was present inthe human B-cell.

In a further embodiment of the present invention the anti-HTT antibody,HTT-binding fragment, synthetic or biotechnological variant thereof canbe optimized to have appropriate binding affinity to the target andpharmacokinetic properties. Therefore, at least one amino acid in theCDR or variable region, which is prone to modifications selected fromthe group consisting of glycosylation, oxidation, deamination, peptidebond cleavage, iso-aspartate formation and/or unpaired cysteine issubstituted by a mutated amino acid that lack such alteration or whereinat least one carbohydrate moiety is deleted or added chemically orenzymatically to the antibody. Examples for amino acid optimization canbe found in Table VII, wherein antibodies showing primer-inducedalterations are shown. Additional modification optimizing the antibodyproperties are described in Gavel et al., Protein Engineering 3 (1990),433-442 and Helenius et al., Annu. Rev. Biochem. 73 (2004), 1019-1049.

Alternatively, the antibody of the present invention is an antibody orantigen-binding fragment, derivative or variant thereof, which competesfor binding to HTT with at least one of the antibodies having the V_(H)and/or V_(L) region as depicted in FIG. 1.

The antibody with at least one antibody having the V_(H) and/or V_(L)region as depicted in FIG. 1 competing for binding to HTT may be furthercharacterized in a dot blot assay and/or filter retardation, asdescribed in Example 6, 13, 18, 31 and/or 32. Therefore, in oneembodiment of the present invention the antibody binds to HTT,preferably to HTT with an expanded poly-Q tract consisting of 49 (HD49)repeats in a dot blot assay and/or filter retardation.

Experimental results provided in FIGS. 3, 7, 11, 21, 22 as well as FIGS.32 and 33, and Examples 6, 7, 13, 18, 26 and 32 suggest that some of theanti-HTT antibodies of the present invention preferentially bind todisease causing mutated and/or aggregated forms of human anti-HTT overthe other amyloid forming proteins. In one embodiment thus, the antibodyof the present invention preferentially recognizes mutated and/oraggregated HTT and/or fragment and/or derivatives thereof over otheramyloid forming proteins.

In one embodiment of the present invention the anti-HTT antibody,HTT-binding fragment, synthetic or biotechnological derivative thereofdoes preferentially recognize mutated, aggregated and/or soluble formsof HTT over physiological HTT.

The antibody of the present invention may be human, in particular fortherapeutic applications. Alternatively, the antibody of the presentinvention is a rodent, rodentized or chimeric rodent-human antibody,preferably a murine, murinized or chimeric murine-human antibody or arat, ratinized or chimeric rat-human antibody which are particularlyuseful for diagnostic methods and studies in animals. In one embodimentthe antibody of the present invention is a chimeric rodent-human or arodentized antibody. Furthermore, in one embodiment, the chimericantibody of the present invention, i.e. comprising the variable domainsof a human antibody and generic murine light and heavy constant domainsbind with a high affinity to human HTT. Preferably, the binding affinityof chimeric antibodies is similar to their human counterparts.

In one embodiment the antibody of the present invention is provided bycultures of single or oligoclonal B-cells that are cultured and thesupernatant of the culture which contains antibodies produced by saidB-cells, is screened for presence and affinity of anti-HTT antibodiestherein. The screening process comprises screening for binding to nativemonomeric, fibrillar or non-fibrillar aggregates like oligomers of hHTTderived from a synthetic full-length hHTT peptide or e.g. purified fromhuman plasma or recombinant expression.

As mentioned above, due to its generation upon a human immune responsethe human monoclonal antibody of the present invention will recognizeepitopes which are of particular pathological relevance and which mightnot be accessible or less immunogenic in case of immunization processesfor the generation of, for example, mouse monoclonal antibodies and invitro screening of phage display libraries, respectively. Accordingly,it is prudent to stipulate that the epitope of the human anti-HTTantibody of the present invention is unique and no other antibody whichis capable of binding to the epitope recognized by the human monoclonalantibody of the present invention exists, A further indication for theuniqueness of the antibodies of the present invention is the fact that,as indicated in FIGS. 19, 20, 24, and 27 to 29, antibodies of thepresent invention bind epitopes that are specific for the mutated and/oraggregated forms of HTT, which as indicated above, are of particularpathological relevance and may not be obtainable by the usual processesfor antibody generation, such as immunization or in vitro libraryscreenings.

Therefore, in one embodiment the present invention also extendsgenerally to anti-HTT antibodies and HTT-binding molecules which competewith the human monoclonal antibody of the present invention for specificbinding to HTT. The present invention is more specifically directed toan antibody, or antigen-binding fragment, variant or derivativesthereof, where the antibody specifically binds to the same epitope in apolyP-region of HTT as a reference antibody selected from the groupconsisting of NI-302.33C11, NI-302.74C11, NI-302.15F9, NI-302.39G12,NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8,NI-302.7A8, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1,NI-302.8F1, NI-302.52C9, NI-302.78H12 and NI-302.46C9. Further, in oneembodiment the present invention is more specifically directed to anantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody specifically binds to the same epitope in theP-rich-region of HTT as a reference antibody selected from the groupconsisting of NI-302.63F3, NI-302.31F11, NI-302.2A2, NI-302.15D3 and/orNI-302.64E5. In another embodiment the present invention is directed toan antibody, or antigen-binding fragment, variant or derivativesthereof, which binds to the same epitope in the C-terminal-region of HTTas a reference antibody selected from the group consisting ofNI-302.35C1 and/or NI.302-72F10. In a further embodiment the presentinvention is directed to an antibody, or antigen-binding fragment,variant or derivatives thereof, which binds to the same epitope in theN-terminal-region of HTT as a reference antibody selected from the groupconsisting of NI-302.15E8. In another embodiment the present inventionis directed to an antibody, or antigen-binding fragment, variant orderivatives thereof, which binds to the same epitope of HTT as areference antibody selected from the group consisting of NI-302.6N9,NI-320.12H2, NI-302.8M1 and/or NI-302.4A6. In one embodiment the presentinvention is directed to an antibody, or antigen-binding fragment,variant or derivatives thereof, which binds to the same epitope of HTTas reference antibody NI-302.7D8. In a preferred embodiment the presentinvention also extends generally to anti-HTT antibodies and HTT-bindingmolecules which compete with the human monoclonal antibody of thepresent invention for specific binding to mutated and/or aggregated HTTspecies or fragments thereof, as shown in Examples 7, 13, 18, 31 and 33as well as FIGS. 7, 13, 19 and 31. The present invention is therefore,more specifically also directed to an antibody, or antigen-bindingfragment, variant or derivatives thereof, where the antibodyspecifically binds to the same epitope in a polyP-region of mutatedand/or aggregated HTT species or fragments thereof as a referenceantibody selected from the group consisting of NI-302.74C11,NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.37C12,NI-302.55D8, NI-302.78H12, NI-302.71F6, NI-302.33C11, NI-302.44D7,NI-302.7A8, NI-302.3D8, NI-302.46C9, NI-302.11H6, NI-302.18A1,NI-302.52C9, and/or NI-302.8F1. Further, in one embodiment the presentinvention is more specifically directed to an antibody, orantigen-binding fragment, variant or derivatives thereof, where theantibody specifically binds to the same epitope in the P-rich-region ofmutated and/or aggregated HTT species or fragments thereof as areference antibody selected from the group consisting of NI-302.63F3,NI-302.31F11, NI-302.2A2, NI-302.15D3 and/or NI-302.64E5. In anotherembodiment the present invention is directed to an antibody, orantigen-binding fragment, variant or derivatives thereof, which binds tothe same epitope in the C-terminal-region of mutated and/or aggregatedHTT species or fragments thereof as a reference antibody selected fromthe group consisting of NI-302.35C1 and/or NI.302-72F10. In a furtherembodiment the present invention is directed to an antibody, orantigen-binding fragment, variant or derivatives thereof, which binds tothe same epitope in the N-terminal-region of mutated and/or aggregatedHTT species or fragments thereof as a reference antibody selected fromthe group consisting of NI-302.15E8. In another embodiment the presentinvention is directed to an antibody, or antigen-binding fragment,variant or derivatives thereof, which binds to the same epitope of HTTas a reference antibody selected from the group consisting ofNI-302.6N9, NI-320.12H2, NI-302.8M1 and/or NI-302.4A6. In one embodimentthe present invention is directed to an antibody, or antigen-bindingfragment, variant or biotechnological derivative thereof, which binds tothe same epitope of HTT as reference antibody NI-302.7D8.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as HTT. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay; see Stahli et al.,Methods in Enzymology 9 (1983), 242-253; solid phase directbiotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986),3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid phasedirect labeled assay, solid phase direct labeled sandwich assay; seeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress (1988); solid phase direct label RIA using I¹²⁵ label; see Morelet al., Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand.J. Immunol. 32 (1990), 77-82. Typically, such an assay involves the useof purified HTT or mutated and/or aggregated HTT, such as oligomersand/or fibrils thereof bound to a solid surface or cells bearing eitherof these, an unlabeled test immunoglobulin and a labeled referenceimmunoglobulin, i.e. the human monoclonal antibody of the presentinvention. Competitive inhibition is measured by determining the amountof label bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Preferably, the competitive binding assay is performed under conditionsas described for the ELISA assay in the appended Examples. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50% or 75%. Hence, the present invention is further drawn to anantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody competitively inhibits a reference antibody selectedfrom the group consisting of NI-302.33C11, NI-302.63F3, NI-302.35C1,NI-302.31F11, NI-302.2A2, NI-302.6N9, NI-302.74C11, NI-302.15F9,NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12,NI-302.55D8, NI-302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6,NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9,NI-302.15E8, NI-302.64E5, NI-302.7D8, NI-302.72F10, NI-302.12H2,NI-302.8M1 and/or NI-302.4A6 from binding to HTT.

The present invention is further drawn to an antibody, orantigen-binding fragment, variant or derivatives thereof, where theantibody competitively inhibits a reference antibody selected from thegroup consisting of NI-302.74C11, NI-302.15F9, NI-302.39G12,NI-302.11A4, NI-302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12,NI-302.71F6, NI-302.33C11, NI-302.44D7, NI-302.7A8, NI-302.3D8,NI-302.46C9, NI-302.11H6, NI-302.18A1, NI-302.52C9, NI-302.8F1,NI-302.63F3, NI-302.31F11, NI-302.2A2, NI302.15D3, NI-302.35C1,NI-302.6N9, NI-302.7D8 and/or NI-302.72F10 from binding to mutatedand/or aggregated HTT species or fragments thereof.

In a preferred embodiment the antibody, the binding of an antibody,binding fragment, synthetic or biotechnological variant thereof, to HTT,preferably to HTT with an expanded poly-Q tract consisting of 49 (HD49)repeats can be measured in a dot blot assay and/or filter retardation asdescribed in the Examples, in particular in 7, 13, 18, 31 and/or 33.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)), where at least oneof V_(H)-CDRs of the heavy chain variable region or at least two of theV_(H)-CDRs of the heavy chain variable region are at least 80%, 85%, 90%or 95% identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2 orV_(H)-CDR3 amino acid sequences from the antibodies disclosed herein.Alternatively, the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions of theV_(H) are at least 80%, 85%, 90% or 95% identical to reference heavychain V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 amino acid sequences fromthe antibodies disclosed herein. Thus, according to this embodiment aheavy chain variable region of the invention has V_(H)-CDR1, V_(H)-CDR2and V_(H)-CDR3 polypeptide sequences related to the groups shown in FIG.1 respectively. While FIG. 1 shows V_(H)-CDRs defined by the Kabatsystem, other CDR definitions, e.g., V_(H)-CDRs defined by the Chothiasystem, are also included in the present invention, and can be easilyidentified by a person of ordinary skill in the art using the datapresented in FIG. 1.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIG. 1 respectively.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIG. 1 respectively, except for one, two, three, four, five, orsix amino acid substitutions in any one V_(H)-CDR. In certainembodiments the amino acid substitutions are conservative.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)), where at least oneof the V_(L)-CDRs of the light chain variable region or at least two ofthe V_(L)-CDRs of the light chain variable region are at least 80%, 85%,90% or 95% identical to reference light chain V_(L)-CDR1, V_(L)-CDR2 orV_(L)-CDR3 amino acid sequences from antibodies disclosed herein.Alternatively, the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions of theV_(L) are at least 80%, 85%, 90% or 95% identical to reference lightchain V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 amino acid sequences fromantibodies disclosed herein. Thus, according to this embodiment a lightchain variable region of the invention has V_(L)-CDR1, V_(L)-CDR2 andV_(L)-CDR3 polypeptide sequences related to the polypeptides shown inFIG. 1 respectively. While FIG. 1 shows V_(L)-CDRs defined by the Kabatsystem, other CDR definitions, e.g., V_(L)-CDRs defined by the Chothiasystem, are also included in the present invention.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)) in which theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groupsshown in FIG. 1 respectively. In another embodiment, the presentinvention provides an isolated polypeptide comprising, consistingessentially of, or consisting of an immunoglobulin light chain variableregion (V_(L)) in which the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3regions have polypeptide sequences which are identical to theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groups shown in FIG. 1respectively, except for one, two, three, four, five, or six amino acidsubstitutions in any one V_(L)-CDR. In certain embodiments the aminoacid substitutions are conservative.

An immunoglobulin or its encoding cDNA may be further modified. Thus, ina further embodiment the method of the present invention comprises anyone of the step(s) of producing a chimeric antibody, murinized antibody,single-chain antibody, Fab-fragment, bi-specific antibody, fusionantibody, labeled antibody or an analog of any one of those.Corresponding methods are known to the person skilled in the art and aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor (1988). When derivatives of saidantibodies are obtained by the phage display technique, surface plasmonresonance as employed in the BIAcore system can be used to increase theefficiency of phage antibodies which bind to the same epitope as that ofany one of the antibodies described herein (Schier, Human AntibodiesHybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995),7-13). The production of chimeric antibodies is described, for example,in international application WO 89/09622. Methods for the production ofhumanized antibodies are described in, e.g., European application EP-A10 239 400 and international application WO 90/07861. Further sources ofantibodies to be utilized in accordance with the present invention areso-called xenogeneic antibodies. The general principle for theproduction of xenogeneic antibodies such as human-like antibodies inmice is described in, e.g., international applications WO 91/10741, WO94/02602, WO 96/34096 and WO 96/33735. As discussed above, the antibodyof the invention may exist in a variety of forms besides completeantibodies; including, for example, Fv, Fab and F(ab)₂, as well as insingle chains; see e.g. international application WO 88/09344. In oneembodiment therefore, the antibody of the present invention is provided,which is selected from the group consisting of a single chain Fvfragment (scFv), a F(ab′) fragment, a F(ab) fragment, and a F(ab′)₂fragment.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus; see, e.g., international application WO00/30680 for corresponding technical details.

The antibodies of the present invention may also include additionalmodifications which optimize their therapeutic potential. Thesemodifications comprise but are not limited to modifications to the aminoacid sequence of the antibody (e.g., the variable regions) andpost-translational modifications. Post-translational modifications(PTMs) are chemical modifications that play a key role in functionalproteomics, because they regulate activity, localization and interactionwith other cellular molecules such as proteins, nucleic acids, lipids,and cofactors. Therefore, the optimization of the antibodies may provideseveral advantages such as an improved stability during storage as wellas pharmacokinetics and/or pharmacodynamics profile such as the in vivoor in vitro circulating time of the antibody, increased solubility,stability, increased affinity to the target, decreased off-rate, animproved effector function of the constant region (Fc region) and safetyprofile of the antibody, such as a decreased immunogenicity, or reducedsusceptibility to posttranslational modifications, as shown e.g. inIgawa et al., MAbs 3 (2011), 243-52. Accordingly, in one embodiment ofthe present invention the anti-HTT antibody, HTT-binding fragment,synthetic or biotechnological variant thereof can be optimized, whereinat least one amino acid in the CDR or variable region, which is prone tomodifications including but are not limited to acetylation, acylation,ADP-ribosylation, amidation, deamidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, isomerization,demethylation, formation of covalent cross-links, formation of cysteine,formation of pyroglutamate, formylation, 7-carboxylation, glycosylation,GPI anchor formation, hydroxylation, hydrolysis, iodination,methylation, myristoylation, oxidation, pegylation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, e.g., Creighton,“Proteins: Structures and Molecular Properties,” 2nd eds., Freeman andCo., N.Y., 1992; “Postranslational Covalent Modification of Proteins,”Johnson, eds., Academic Press, New York, 1983; Seifter et al., Meth.Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY. Acad. Sei. 663(1992) 48-62) is substituted by a mutated amino acid that lack suchalteration or wherein at least one carbohydrate moiety is deleted oradded chemically or enzymatically to the antibody. In a preferredembodiment the modifications are selected from the group consisting ofglycosylation, oxidation, deamination, peptide bond cleavage,iso-aspartate formation and/or unpaired cysteine. Additionalmodification that optimize the utility of the HTT-antibodies or bindingmolecules as a therapeutic agent are well known in the art and describede.g. in Igawa et al., MAbs 3 (2011), 243-52 which disclosure content isincorporated herein. Means of adding or deleting carbohydrate moietiescan be achieved chemically or enzymatically and is described in detailin e.g. Berg et al. “Biochemistry” 5th eds W H Freeman, New York 2002;WO 87/05330; Aplin et al., CRC Crit. Rev. Biochem., 22 (1981), 259-306;Hakimuddin et al., Arch. Biochem. Biophys., 259 (1987), 10-52; Edge etal., Anal. Biochem., 118 (1981), 131; Thotakura et al., Meth. Enzymol.138. (1987), 350.

Additionally, the present invention encompasses peptides including thosecontaining a binding molecule as described above, for example containingthe CDR3 region of the variable region of any one of the mentionedantibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that heavy chain CDR3 (HCDR3) is the regionhaving a greater degree of variability and a predominant participationin antigen-antibody interaction. Such peptides may easily be synthesizedor produced by recombinant means to produce a binding agent usefulaccording to the invention. Such methods are well known to those ofordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can also be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

Hence, the present invention relates to any binding molecule, e.g., anantibody or binding fragment thereof which is oriented towards theanti-HTT antibodies and/or antibodies capable of binding mutated and/oraggregated HTT species and/or fragments thereof of the present inventionand displays the mentioned properties, i.e. which specificallyrecognizes HTT and/or mutated and/or aggregated HTT species and/orfragments thereof. Such antibodies and binding molecules can be testedfor their binding specificity and affinity by ELISA andimmunohistochemistry as described herein, see, e.g., the Examples. Thesecharacteristics of the antibodies and binding molecules can be tested byWestern Blot as well.

As an alternative to obtaining immunoglobulins directly from the cultureof B cells or memory B cells, the cells can be used as a source ofrearranged heavy chain and light chain loci for subsequent expressionand/or genetic manipulation. Rearranged antibody genes can be reversetranscribed from appropriate mRNAs to produce cDNA. If desired, theheavy chain constant region can be exchanged for that of a differentisotype or eliminated altogether. The variable regions can be linked toencode single chain Fv regions. Multiple Fv regions can be linked toconfer binding ability to more than one target or chimeric heavy andlight chain combinations can be employed. Once the genetic material isavailable, design of analogs as described above which retain both theirability to bind the desired target is straightforward. Methods for thecloning of antibody variable regions and generation of recombinantantibodies are known to the person skilled in the art and are described,for example, Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doeneckeet al., Leukemia 11 (1997), 1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, however, mammaliancells are preferred. Typical mammalian cell lines useful for thispurpose include, but are not limited to, CHO cells, HEK 293 cells, orNSO cells.

The production of the antibody or analog is then undertaken by culturingthe modified recombinant host under culture conditions appropriate forthe growth of the host cells and the expression of the coding sequences.The antibodies are then recovered by isolating them from the culture.The expression systems are preferably designed to include signalpeptides so that the resulting antibodies are secreted into the medium;however, intracellular production is also possible.

In accordance with the above, the present invention also relates to apolynucleotide encoding the antibody or equivalent binding molecule ofthe present invention, in case of the antibody preferably at least avariable region of an immunoglobulin chain of the antibody describedabove. Typically, said variable region encoded by the polynucleotidecomprises at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) of the variable region of the said antibody.

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat; see, e.g., Riechmann, et al, Nature 332 (1988),323-327. Thus, the present invention also relates to antibodies whereinone or more of the mentioned CDRs comprise one or more, preferably notmore than two amino acid substitutions. Preferably, the antibody of theinvention comprises in one or both of its immunoglobulin chains two orall three CDRs of the variable regions as set forth in FIG. 1.

Binding molecules, e.g., antibodies, or antigen-binding fragments,synthetic or biotechnological variants, or derivatives thereof of theinvention, as known by those of ordinary skill in the art, can comprisea constant region which mediates one or more effector functions. Forexample, binding of the C1 component of complement to an antibodyconstant region may activate the complement system. Activation ofcomplement is important in the opsonization and lysis of cell pathogens.The activation of complement also stimulates the inflammatory responseand may also be involved in autoimmune hypersensitivity. Further,antibodies bind to receptors on various cells via the Fc region, with aFc receptor binding site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

Accordingly, certain embodiments of the present invention include anantibody, or antigen-binding fragment, variant, or derivative thereof,in which at least a fraction of one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of HTT aggregation and deposition, reduced serum half-life, orincreased serum half-life when compared with a whole, unaltered antibodyof approximately the same immunogenicity. For example, certainantibodies for use in the diagnostic and treatment methods describedherein are domain deleted antibodies which comprise a polypeptide chainsimilar to an immunoglobulin heavy chain, but which lack at least aportion of one or more heavy chain domains. For instance, in certainantibodies, one entire domain of the constant region of the modifiedantibody will be deleted, for example, all or part of the CH2 domainwill be deleted. In other embodiments, certain antibodies for use in thediagnostic and treatment methods described herein have a constantregion, e.g., an IgG heavy chain constant region, which is altered toeliminate glycosylation, referred to elsewhere herein as aglycosylatedor “agly” antibodies. Such “agly” antibodies may be preparedenzymatically as well as by engineering the consensus glycosylationsite(s) in the constant region. While not being bound by theory, it isbelieved that “agly” antibodies may have an improved safety andstability profile in vivo. Methods of producing aglycosylatedantibodies, having desired effector function are found for example ininternational application WO 2005/018572, which is incorporated byreference in its entirety.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing HTT localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half-life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as HTTlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences to increase the cellularuptake of antibodies by way of example by enhancing receptor-mediatedendocytosis of antibodies via Fcγ receptors, LRP, or Thy1 receptors orby ‘SuperAntibody Technology’, which is said to enable antibodies to beshuttled into living cells without harming them (Expert Opin. Biol.Ther. (2005), 237-241). For example, the generation of fusion proteinsof the antibody binding region and the cognate protein ligands of cellsurface receptors or bi- or multi-specific antibodies with a specificsequences binding to HTT as well as a cell surface receptor may beengineered using techniques known in the art.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences or the antibody may bechemically modified to increase its blood brain barrier penetration.Modified forms of antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be made from whole precursor orparent antibodies using techniques known in the art. Exemplarytechniques are discussed in more detail herein. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be made or manufactured using techniques that are known inthe art. In certain embodiments, antibody molecules or fragments thereofare “recombinantly produced”, i.e., are produced using recombinant DNAtechnology. Exemplary techniques for making antibody molecules orfragments thereof are discussed in more detail elsewhere herein.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention also include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In particular preferred embodiments, antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention will notelicit a deleterious immune response in the animal to be treated, e.g.,in a human. In certain embodiments, binding molecules, e.g., antibodies,or antigen-binding fragments thereof of the invention are derived from apatient, e.g., a human patient, and are subsequently used in the samespecies from which they are derived, e.g., human, alleviating orminimizing the occurrence of deleterious immune responses.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes; see, e.g., internationalapplications WO 98/52976 and WO 00/34317. For example, V_(H) and V_(L)sequences from the starting antibody are analyzed and a human T cellepitope “map” from each V region showing the location of epitopes inrelation to complementarity determining regions (CDRs) and other keyresidues within the sequence. Individual T cell epitopes from the T cellepitope map are analyzed in order to identify alternative amino acidsubstitutions with a low risk of altering activity of the finalantibody. A range of alternative V_(H) and V_(L) sequences are designedcomprising combinations of amino acid substitutions and these sequencesare subsequently incorporated into a range of binding polypeptides,e.g., HTT-specific antibodies or immunospecific fragments thereof foruse in the diagnostic and treatment methods disclosed herein, which arethen tested for function. Typically, between 12 and 24 variantantibodies are generated and tested. Complete heavy and light chaingenes comprising modified V and human C regions are then cloned intoexpression vectors and the subsequent plasmids introduced into celllines for the production of whole antibody. The antibodies are thencompared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981), said references incorporatedby reference in their entireties. The term “monoclonal antibody” as usedherein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. In certain embodiments, antibodies of the presentinvention are derived from human B cells which have been immortalizedvia transformation with Epstein-Barr virus, as described herein.

In the well-known hybridoma process (Kohler et al., Nature 256 (1975),495) the relatively short-lived, or mortal, lymphocytes from a mammal,e.g., B cells derived from a human subject as described herein, arefused with an immortal tumor cell line (e.g., a myeloma cell line),thus, producing hybrid cells or “hybridomas” which are both immortal andcapable of producing the genetically coded antibody of the B cell. Theresulting hybrids are segregated into single genetic strains byselection, dilution, and re-growth with each individual straincomprising specific genes for the formation of a single antibody. Theyproduce antibodies, which are homogeneous against a desired antigen and,in reference to their pure genetic parentage, are termed “monoclonal”.

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. The binding specificity of the monoclonal antibodiesproduced by hybridoma cells is determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA) as described herein. After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods; see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press (1986),59-103. It will further be appreciated that the monoclonal antibodiessecreted by the subclones may be separated from culture medium, ascitesfluid or serum by conventional purification procedures such as, forexample, protein-A, hydroxylapatite chromatography, gel electrophoresis,dialysis or affinity chromatography.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized or naturally immunemammal, e.g., a human, and cultured for about 7 days in vitro. Thecultures can be screened for specific IgGs that meet the screeningcriteria. Cells from positive wells can be isolated. IndividualIg-producing B cells can be isolated by FACS or by identifying them in acomplement-mediated hemolytic plaque assay. Ig-producing B cells can bemicromanipulated into a tube and the V_(H) and V_(L) genes can beamplified using, e.g., RT-PCR. The V_(H) and V_(L) genes can be clonedinto an antibody expression vector and transfected into cells (e.g.,eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments may be producedrecombinantly or by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)₂ fragments). F(ab′)₂ fragments contain the variableregion, the light chain constant region and the CH1 domain of the heavychain. Such fragments are sufficient for use, for example, inimmunodiagnostic procedures involving coupling the immunospecificportions of immunoglobulins to detecting reagents such as radioisotopes.

In one embodiment, an antibody of the invention comprises at least oneCDR of an antibody molecule. In another embodiment, an antibody of theinvention comprises at least two CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least three CDRs from one or more antibody molecules. In anotherembodiment, an antibody of the invention comprises at least four CDRsfrom one or more antibody molecules. In another embodiment, an antibodyof the invention comprises at least five CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least six CDRs from one or more antibody molecules. Exemplaryantibody molecules comprising at least one CDR that can be included inthe subject antibodies are described herein.

Antibodies of the present invention can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably by recombinant expression techniques asdescribed herein.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ΔCH2 constructs). Forother embodiments a short connecting peptide may be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the CH2 domain on the catabolic rate of the antibody. Domain deletedconstructs can be derived using a vector encoding an IgG₁ human constantdomain, see, e.g., international applications WO 02/060955 and WO02/096948A2. This vector is engineered to delete the CH2 domain andprovide a synthetic vector expressing a domain deleted IgG₁ constantregion.

In certain embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the present invention areminibodies. Minibodies can be made using methods described in the art,see, e.g., U.S. Pat. No. 5,837,821 or international application WO94/09817.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises an immunoglobulin heavychain having deletion or substitution of a few or even a single aminoacid as long as it permits association between the monomeric subunits.For example, the mutation of a single amino acid in selected areas ofthe CH2 domain may be enough to substantially reduce Fc binding andthereby increase HTT localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement binding) to be modulated.Such partial deletions of the constant regions may improve selectedcharacteristics of the antibody (serum half-life) while leaving otherdesirable functions associated with the subject constant region domainintact. Moreover, as alluded to above, the constant regions of thedisclosed antibodies may be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as an effector function or provide for morecytotoxin or carbohydrate attachment. In such embodiments it may bedesirable to insert or replicate specific sequences derived fromselected constant region domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the V_(H) regions and/or V_(L) regions)described herein, which antibodies or fragments thereofimmunospecifically bind to HTT. Standard techniques known to those ofskill in the art can be used to introduce mutations in the nucleotidesequence encoding an antibody, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions.

Preferably, the variants (including derivatives) encode less than 50amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference V_(H) region, V_(H)-CDR1, V_(H)-CDR2,V_(H)-CDR3, V_(L) region, V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind HTT and/or mutated and/or aggregated HTT species and/orfragments thereof).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, e.g., have no, orlittle, effect on an antibody's ability to bind antigen, indeed somesuch mutations do not alter the amino acid sequence whatsoever. Thesetypes of mutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Codon-optimized coding regions encodingantibodies of the present invention are disclosed elsewhere herein.Alternatively, non-neutral missense mutations may alter an antibody'sability to bind antigen. The location of most silent and neutralmissense mutations is likely to be in the framework regions, while thelocation of most non-neutral missense mutations is likely to be in CDR,though this is not an absolute requirement. One of skill in the artwould be able to design and test mutant molecules with desiredproperties such as no alteration in antigen-binding activity oralteration in binding activity (e.g., improvements in antigen-bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of HTT and/or mutatedand/or aggregated HTT species and/or fragments thereof) can bedetermined using techniques described herein or by routinely modifyingtechniques known in the art.

III. Polynucleotides Encoding Antibodies

A polynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxribonucleotide, which may be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding an antibody,or antigen-binding fragment, variant, or derivative thereof can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single-stranded anddouble-stranded regions.

In addition, a polynucleotide encoding an antibody, or antigen-bindingfragment, variant, or derivative thereof can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

As is well known, RNA may be isolated from the original B cells,hybridoma cells or from other transformed cells by standard techniques,such as a guanidinium isothiocyanate extraction and precipitationfollowed by centrifugation or chromatography. Where desirable, mRNA maybe isolated from total RNA by standard techniques such as chromatographyon oligo dT cellulose. Suitable techniques are familiar in the art. Inone embodiment, cDNAs that encode the light and the heavy chains of theantibody may be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well-known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as human constant region probes. DNA, typicallyplasmid DNA, may be isolated from the cells using techniques known inthe art, restriction mapped and sequenced in accordance with standard,well known techniques set forth in detail, e.g., in the foregoingreferences relating to recombinant DNA techniques. Of course, the DNAmay be synthetic according to the present invention at any point duringthe isolation process or subsequent analysis.

In this context, the present invention also relates to a polynucleotideencoding at least the binding domain or variable region of animmunoglobulin chain of the antibody of the present invention. In oneembodiment, the present invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin heavy chain variable region (V_(H)), where atleast one of the CDRs of the heavy chain variable region or at least twoof the V_(H)-CDRs of the heavy chain variable region are at least 80%,85%, 90%, or 95% identical to reference heavy chain V_(H)-CDR1,V_(H)-CDR2, or V_(H)-CDR3 amino acid sequences from the antibodiesdisclosed herein. Alternatively, the V_(H)-CDR1, V_(H)-CDR2, orV_(H)-CDR3 regions of the V_(H) are at least 80%, 85%, 90%, or 95%identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2, andV_(H)-CDR3 amino acid sequences from the antibodies disclosed herein.Thus, according to this embodiment a heavy chain variable region of theinvention has V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIG. 1.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(V_(L)), where at least one of the V_(L)-CDRs of the light chainvariable region or at least two of the V_(L)-CDRs of the light chainvariable region are at least 80%, 85%, 90%, or 95% identical toreference light chain V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 amino acidsequences from the antibodies disclosed herein. Alternatively, theV_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 regions of the V_(L) are at least80%, 85%, 90%, or 95% identical to reference light chain V_(L)-CDR1,V_(L)-CDR2, and V_(L)-CDR3 amino acid sequences from the antibodiesdisclosed herein. Thus, according to this embodiment a light chainvariable region of the invention has V_(L)-CDR1, V_(L)-CDR2, orV_(L)-CDR3 polypeptide sequences related to the polypeptide sequencesshown in FIG. 1.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(V_(H)) in which the V_(H)-CDR1, V_(H)-CDR2, and V_(H)-CDR3 regions havepolypeptide sequences which are identical to the V_(H)-CDR1, V_(H)-CDR2,and V_(H)-CDR3 groups shown in FIG. 1.

As known in the art, “sequence identity” between two polypeptides or twopolynucleotides is determined by comparing the amino acid or nucleicacid sequence of one polypeptide or polynucleotide to the sequence of asecond polypeptide or polynucleotide. When discussed herein, whether anyparticular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2 (1981), 482-489, to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

In a preferred embodiment of the present invention, the polynucleotidecomprises, consists essentially of, or consists of a nucleic acid havinga polynucleotide sequence of the V_(H) or V_(L) region of an anti-HTTantibody and/or antibody recognizing a polyP-region in the HTT and/ormutated and/or aggregated HTT species and/or fragments thereof asdepicted in and Table II. Additionally, in one embodiment thepolynucleotide comprises, consists essentially of, or consists of anucleic acid having a polynucleotide sequence of the V_(H) or V_(L)region of an anti-HTT antibody and/or antibody recognizing theP-rich-region in the HTT and/or mutated and/or aggregated HTT speciesand/or fragments thereof as depicted in and Table III and/or furtherrecognizing the C-terminal region in the HTT and/or mutated and/oraggregated HTT species and/or fragments thereof as depicted in and TableIV and/or further recognizing the Q/P-rich region of HTT and/or mutatedand/or aggregated HTT species and/or fragments thereof as depicted inTable VII. In addition, in one embodiment the polynucleotide comprises,consists essentially of, or consists of a nucleic acid having apolynucleotide sequence of the V_(H) or V_(L) region of an anti-HTTantibody and/or antibody recognizing the HTT and/or mutated and/oraggregated HTT species and/or fragments thereof as depicted in and TableV. Furthermore, in one embodiment the polynucleotide comprises, consistsessentially of, or consists of a nucleic acid having a polynucleotidesequence of the V_(H) or V_(L) region of an anti-HTT antibody and/orantibody recognizing the N-terminal region in the HTT and/or mutatedand/or aggregated HTT species and/or fragments thereof as depicted inand Table VI. In addition, in one embodiment the polynucleotidecomprises, consists essentially of, or consists of a nucleic acid havinga polynucleotide sequence of the V_(H) or V_(L) region of an anti-HTTantibody and/or antibody recognizing the HTT and/or mutated and/oraggregated HTT species and/or fragments thereof as depicted in and TableV. Additionally or alternatively, in one embodiment the polynucleotidecomprises, consists essentially of, or consists of a nucleic acid havinga polynucleotide sequence of the V_(H) or V_(L) region of an anti-HTTantibody and/or antibody as depicted in and Table VIII.

In this respect, the person skilled in the art will readily appreciatethat the polynucleotides encoding at least the variable domain of thelight and/or heavy chain may encode the variable domain of bothimmunoglobulin chains or only one. In one embodiment therefore, thepolynucleotide comprises, consists essentially of, or consists of anucleic acid having a polynucleotide sequence of the V_(H) and the V_(L)region of an anti-HTT antibody and/or fragments thereof as depicted inTable II, III, IV, V, VI, VII or VIII.

TABLE IINucleotide sequences of the V_(H) and V_(L) region of antibodies recognizing anepitope of a polyP-region of HTT, i.e exon 1 in aggregated form.Nucleotide sequences of variable Antibodyheavy (V_(H)) and variable light (V_(L)) chains NI-302.33C11-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTTGTCCAGCCTGGGAACTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCAGGTTCAGTGACTTTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGCTGGCACTTATATGGTATGATGGAGGGTATAAGTACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGATGTTTCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGACCCACCTAGAATATTGCAGTAGAACCACCTGCTATCTCGGCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCC TCGSEQ ID NO: 1 NI-302.33C11-V_(K)GACATCCAGTTGACCCAGTCTCCGTCCTTCCTATCTGCGTCTGTGGGAGACACAGTCACCTTCACTTGCCGGGCCAGTCAGGGCATTAGCGATTATTTAGCCTGGTTTCAGCAGAAACCAGGGATTGCCCCTAAGCTCCTGATCTATGCTGCGTCCACTTTGCAAACCGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCCGCAGCCTGCAGTCTGAAGATTTTGGAACTTATTACTGTCAGCAGCTTAAAACTTACCCGTACACTTTTGGCCAGGGGACCAAGGTGGAAATCAAA SEQ ID NO: 3 NI-302.74C11-V_(H)GAGGTGCAGCTGGTGCAGTCTGGGACTGAGGTGCAGAAGCCTGGGGCCTCAGTAAAAGTCTCCTGCAAGGCTTCTGGATACAGTTTCACCGGCTACTTTTTGCACTGGGTACGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGTGGATCAACCCTAACAGTGGTGACACAAACTATGCAGAGAAGTTTCGGGGCAGAATCATCATGACCAGGGACACGTCTGTCAGCACAGCCCACATGGAGTTGAGCAGCCTGAGATTTGACGACACGGCCCTATATTACTGTACGAGAGAGGCCCCTGACCCGGGCGCTGAGACGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 25NI-302.74C11-V_(L)CAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCTGGAGATGCAGTGCCAAAGCAGTATATTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTATTCTGGTGATATATAAAGACACTCAGAGGCCTTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCAGGGACAACAGTCACGTTGACCATAACTGGCGTCCAGGCAGACGACGAGGGTGACTATTACTGTCAATCAGCAGACAGTAGTGCTACTTGGGTGTTCGGCGGAGGGACCAAATTGACCGTCCTA SEQ ID NO: 27 NI-302.15F9-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCACGCCGGGGGGGTCCCTGAGACTCTCGTGTGAGGCCTCTGGATTTCTCTTCAAGAATTCTAGCATGAACTGGGTCCGTCAGACTCCGGGGAAGGGGCTGGAGTGGGTCTCGTCCATTGACACTTCTGCTACAAATTATAAGTATTATGCAGACTCTGTGAAGGGCCGATTTACCATCTCCAGGGATGACGCCACCAACTCTCTCTATCTGCAAATGAATAGCCTGCGAGCCGACGACACGGCTACTTATTACTGTGCGCGAGGTTATTATACCCCCCGGGACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 29NI-302.15F9-V_(K)GATGTTGTGATGACTCAGTCTCCACAGACCCTGTCCGTCAGCCTTGGACAGGCGGCCTCCATCTCCTGCAGGTCGAGTCAAAGCCTCTTGTATCGTGATAACAACACATACTTGAATTGGTTTCACCAGAGGCCAGGCCAATCTCCAAGGCGCCTCATTTATAGGGCTTCTGACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCGGTGGGTCAGGCACTGATTTCACATTGAAAATCAGTGGAGTGGAGGCTGAAGATGTTGGCACTTATTACTGCATGCAAGGAACACACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 31 NI-302.39G12-V_(H)GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCACCCTTGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCGTCTCTAATTACGCCATAACTTGGGTCCGCCGGGCTCCAGGGAAGGGGCTGCAATATATTTCAGTAATTTATCGTGATGGCAGGACATACTACGGAGACTCCGTGAGGGGCCGCTTCACCATCTCTAGGGACGATTCCAAGAACACTCTCTATCTTCAAATGAACAGCCTGAGATTTGAGGACACGGCTGTGTATTACTGTGCGAGAGCGCACGGCCAATATTACTATGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 33NI-302.39G12-V_(K)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCAGCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATAGTAATGGATACAACTATTTGGATTGGTACCGGCAGAAACCAGGGCAGTCTCCACAGCTCCTGATCTATTTGAGTTCTAATCGGCCCTCCGGGGTCCCTGATAGGTTCAGTGCCAGTGGATCAGGCACAGAGTTCACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCGGAGGGACCAAAGTGGATATCAAA SEQ ID NO: 35 NI-302.11A4-V_(H)GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCCCCGTCAGTAGCAGTTACATGAGCTGGGTCCGCCAGGCTCCAGGAGAGGGGCTGGAGTGGGTCTCAGTTCTTTATAGAGACGGTGACACATACTACGCAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAATTCCCAGAACACGTTCTATCTTCAAATGAACAGCCTGAAAGCCGAGGACACGGCCGTGTATTACTGTGCGGGTGATAGAAGGTCGTCACACTACTATTACGGTATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 37NI-302.11A4-V_(K)GAAATTGTGATGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGAGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTCGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTACGTCCCGCAGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAACAGTATGGTAGCTCGTGGACGTTCGGCCCAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 39 NI-302.22H9-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCACCCTTGGGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCAGCGTCTCTAATTACGCCATAACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATATATTTCAGTGATTTATCGTGATGGCAGGACATACTACGGAGACTCCGTGAGGGGCCGCTTCACCATCTCTAGGGACGATTCCAAGAACACTATCTATCTTCAAATGAACAGCCTGAGATTTGAGGACACGGCTGTGTATTACTGTGCGAGAGCGCACGGCCAATATTATTATGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO 41:NI-302.22H9-V_(K)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCAGCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATAGTAATGGATACAACTATTTGGATTGGTACCGGCAGAAACCAGGGCAGTCTCCACAACTCCTGATCTATTTGAATTCTAATCGGGCCTCCGGGGTCCCTGATAGGTTCAGTGGCAGTGGATCAGGCACAGAGTTCACACTGACAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 43 NI-302.44D7-V_(H)GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTATAGTGATACTAGCACATATTACGCAGACTCCGTGAAGGGCCGCTTCACCGTCTCCAGAGACATTTCCAAGAACACGCTGTATCTGCAAATGAATAGCCTGAGGGCCGAGGACACGGCCGTATATTACTGCGCGAAAGGTACCAGGGACTATTACGGTATGGACGTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO: 45NI-302.44D7-V_(K)CAGACTGTGGTGACTCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTCACTTGTGGCTTGAGTTCTGGCTCAGTTTCTACTAGTTACTACCCCAGCTGGTACCAGCAGACCCCAGGCCGGGCTCCACGCACGCTCATCTACAGCACAAACACTCGCTCTTCTGGGGTCCCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAGGCTGCCCTCACCATCACGGGGGCCCAGGCAGATGATGAATCTGATTATTACTGTGTGCTGTTTATGGGTAGTGGCATTGGGGTGTTCGGCGGAGGGACCAGGCTGACCGTCCTA SEQ ID NO: 47 NI-302.37C12-V_(H)GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCTTGTGTTGCCTCTGCACTCACCGTCACTAACAGCCAAATGACCTGGGTCCGCCGGGCTCCAGGGAGGGGGTTGGAGTGGGTCTCAGTTATTTACACCAGTGGTAGTGCATACTACGCAGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTGAGAGTCGAAGACACGGCTGTGTATTACTGTGCGAAAGGCCCATCAGCCTATTATTACGGTTTGGACCTTTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 49NI-302.37C12-V_(K)GATATTGTGATGACTCAATCACCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCGGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAGACGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 51 NI-302.55D8-V_(H)CAGGTGCAGCTGGTGCAGTCTGGGTCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGACGGATCAACCCTAACAATGGTGGCACAAACTATGCACAGAACTTTCAGGGCTGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTCAGCAGACTGAGATCTGACGACACGGCCGTCTATTACTGTGCGAGAGTGGGGGGCGAGCTGCTACGAGAAGGCGGCTATCACTACTACATGGACGTCTGGGGCAAGGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 53 NI-302.55D8-V_(L)CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAACAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTTTGATAATACCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTAATTATCACTGCCAGTCCTATGACAACAGCCTGAGTGGTTCTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 55 NI-302.7A8-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCATATTTAGAAACAGTTGGATGACCTGGGTCCGCCAGGATCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGGAAGATGGAAGTCGGACATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAGGAGATTATAATTCGGGCATCTATTACTTTCCCGGGGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCC TCGSEQ ID NO: 57 NI-302.7A8-V_(K)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGTAGGTCTAGTCAAAGCCTCGTATACAGTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAGTCTCCAAGGCGCCTCATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAGAATCAGCAGGGTGGAGGCTGAGGATGTTGGCATTTATTACTGCATGCAAGGTACACACTGGCCTGGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 59 NI-302.78H12-V_(H)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGTCTTGTCTCTAGTTACTCCATCAGCAATGGTTACTACTGGGGCTGGATTCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATCATAATGGGAACACCTATTACAACCCGTCCCTCAAGAGTCGAGTCATCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGTTGAGGTCTGTGACCGCCGCAGACACGGCCGTGTACTACTGTGCGATGCCAAGTGCCACCTATTATTATGGTTCGGGGACTCAATTCCATGCGTTTGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 61 NI-302.78H12-V_(L)CAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGAGATGTTGGTAATTATAACTATGTCTCCTGGTACCAACAACACCCAGGCGAAGTCCCCAAACTCATAATTTATGATGTCAGTGAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCGCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGACTATTACTGCTGCTCATATGCTGGCAGTTACACCTTCGAGGTATTTGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 63 NI-302.71F6-V_(H)CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTATTGAAGCCTTCGGAGACCCTGTCCCTCACGTGCGCTGTCTATGGTGGGTCCCTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATAGGGGAAGTCAATCATAGTGGAGGCACCAACCTCAATTCGTCCCTCAAGAGTCGAGTCATCATTTCAGTAGACAAGTCCAAGAAGCAGTTCTCCCTGAAACTGAGCTCTGTGACCGCCGCGGACACGGCTATGTACTTCTGTGCGAGAGGATACAGCTATGACCCAAAATACTACTTTGACTCCTGGAGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 65NI-302.71F6-V_(L)CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGGCGATCACCATCTCCTGCACTGGAACCAGTAGTGATATTGGGAGTTATGATTTTGTCTCCTGGTACCAGCAGGACCCAGGCAAAGCCCCCAAAGTCATTATTTATGGGGTCAATAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGACTCCAGGCTGACGACGAGGCTGATTATTACTGCTGCTCATATGCTGGTAGTACCACTTGGGTGTTCGGCGGAGGGACCAAACTGACCGTCCTA SEQ ID NO: 67 NI-302.11H6-V_(H)GAGGTGCAGCTGGTGCAGTCTGGAGCTGTGATGAAGAAGCCTGGAGACTCAGTGAGGGTCTCCTGCAGGGCTTCTACTTACAGCTTTTCCACCTATAGTTTCACCTGGGTGCGACAGGTCCCTGGACAAGGCCTTGAGTGGATGGGATGGATCAGCGCTTATAATGGTCACACAAACTATGTAGACAGCTTCCAGGGCAGACTCACGTTGACCACAGACACATCCGCGAGTACAGCGTACATGGAACTGAGGAGCCTCAGATCTGACGACACGGCCATCTATTATTGTGCGGCTGTAGACACCACTTACTACTATTACGGCATGGACGTCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 69NI-302.11H6-V_(L)CAGACTGTGGTGACTCAGGAGCCAACGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTCACTTGTGCCTTGAGGTTTGGCTCAGTCTCTAGTAGCTACTATCCCAGCTGGTTCCAGCAGACCCCAGGCCAGGCTCCACGCACGCTCATCTACAGCACAAACACCCGCTCTTCGGGGGTCCCTGCTCGATTCTCTGGCTCCATTCTTGGGAACAAAGCTGCCCTCACCATCGCGGGGGCCCAGGCAAATGATGAGGCTGACTATTACTGTGTGCTGTATATGGGTAGTGGAATCGGGGTGTTCGGCGGAGGGACCAAGTTGACCGTCCTA SEQ ID NO: 71 NI-302.3D8-V_(H)GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCTTGTGAAGCCTCCGGATTCATCTTTAAAACCTATGCCATGAGCTGGGTCCGCCAGCTTCCCGGGAGGGGGCTGGAATGGGTCTCAGCTATAAGTGCCACTGGTGGAAGCACCTTCTACGCAGAGTCCGTGAAGGGCCGGCTCACCATTTCCAGAGACACTGCCAAGAATACAGTGTATCTGCAAATGAACAACCTGAGAGCCGAAGACACGGCCATGTATTACTGTGCGAAAGGGTCGACTGCGGTATATCTCTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 73NI-302.3D8-V_(K)GACATCCAGATGACCCAGTCTCCGTCCTCACTGTCTGCATCTGTAGGGGACAGAGTCACCCTCACTTGTCGGGCGAGTCAGGACATCAGAAATTTCTTGGCCTGGATTCAGCAGAAGCCAGGGAAACCCCCTAAGTCCCTGATCTATGCTGCGTCCACTTTGCAAAGTGGGGTCCCATCACGATTCAGCGGCAGTGGATCCGGGACAGATTTCACTCTCACCATCAGCAGCCTGCACCCTGAAGATTTTGCTACTTATTACTGCCAGCAGTTTTATAATTACCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 75 NI-302.18A1-V_(H)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTAGTGAAGCCTTCGGAGGCCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCACTACTGATTATTACTATTGGGGCTGGATCCGCCAGTCCCCAGGCAAGGGACTAGAGTGGGTTGGGACAATATACTTTGGTGGGGCCACCTACTACAATCCGTCCCTCAGGAACCGGGTCTCGATATCTGTGGACACGTCCAACACTCGCCTCTCCCTGAGACTTATCTCTCTGAGCGCCGCTGACACGGCCGTCTATTATTGTGCGAGAGTGGGCTACTTGGATAGGAGTGGTCTTCTTGTGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 77NI-302.18A1-V_(K)GAAATTGTGCTGACGCAGTCTCCACTCTCCGTGCCCGTCACCCCCGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAATAATGGATACAACTATTTGGATTGGTACCTGAAGAAGCCTGGGCAGTCTCCACAACTCCTGATCTATTTGGGCTCTACTCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGCCAGTGGATCAGGCACAGACTTTACACTGGAAATCAGCAGAGTGGAGGCTGAAGATGTTGGCGTTTACTACTGCATGCAAGCTCTGCAGACTCCTCCGACTTTCGGCAGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 79 NI-302.52C9-V_(H)GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAACCTGGGGGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCGTCAGTGACACCTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTCATGCCGGTGGTGAAACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTTCAAATGAATAGGCTGACACCTGAGGACACGGCTGTCTTTTATTGTGCGAGACACTACTACGGTAATGACGACGACACTGATTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 85NI-302.52C9-V_(K)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACGTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTCATCTATTTGGGTTCTACTCGGGCCTCCGGGGTCCCTGACAGATTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCTTACAAGCTCAACAAATTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 87 NI-302.46C9-V_(H)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTAAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTTTCTGGTGCCTCCGTCAGCAGTGGTGCCTACTACTGGAGTTGGATCCGGCAGCCCGCCGGGAAGCGACTGGAGTGGATTGGGCGTGTCTATCCCACTTGGAGCACCAACTACAACCCCTCCCTCGAGAGTCGAGTCACCATATCGTTAGACACGTCCAACAACCAGTTCTCCCTGAAGCTGACCTCTTTGACTGCCGCAGACACGGCCGTTTATTACTGTGCGAGAGAGGCTCCTGGTGACTACGATGCTGCGCCCCTAGCCTACTGGGGCCAGGGCACCCTGGTCACCGTCTCC TCGSEQ ID NO: 89 NI-302.46C9-V_(K)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGTACATTAGCCACTATTTAAATTGGTATCGGCAGAAACCAGGGAAAGCCCCTCAGCTCGTAATCTATGCTGCATCCAGTTTGCAAAGTGAGGTCCCATCAAGGTTCAGTGGGAGTGGATCTGGGCCAGAGTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGAGTTACACTACCCCTCGAACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 91

TABLE IIINucleotide sequences of the V_(H) and V_(L) region of antibodies recognizing anepitope of the P-rich region of HTT, i.e exon 1 in aggregated form.Nucleotide sequences of variable Antibodyheavy (V_(H)) and variable light (V_(L)) chains NI-302.63F3-V_(H)CAGGTGCAGCTGGTGCAATCTGGGTCTGCGTTCAAGAAGCCTGGGACCTCAGTGAAAGTTTCCTGCAAGGCCTCTGGATACACCTTCGAGACCCGTTCTATGAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATACATGGGATGGATCAACACCAACACTGGCAACCGCACGTATGTCCAGGCCTTCAGAGGACGATTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGATCAGCAACTTAAAGACTGAGGACACTGCCGTGTATTACTGTGCGAGAGGGGCAGGTGGGGGATATTGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 5NI-302.63F3-V_(K)GACATCCAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAATCAGAGTCTTTTCTACAGTTCCAACAATAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 7 NI-302.31F11-V_(H)GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCTTGATCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGCAGCACCTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTTGAGTGCGTCTCAGTTATTTTTAGTGGCGCTGACACATATTACGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAACACACTGTTTCTTCAGATGAACAGCCTGAGAGTCGAGGACACGGCCACATATTACTGTGTGAGACATTATTATGGTTCAGACCTTCCATCTGACTTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 13NI-302.31F11-V_(K)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCGCCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTATACAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGAAGCCTCCACAGCTCCTGGTCTATTTGGGTTCTGATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCAAAGATTTTACACTGAACATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCGTGGACGTTCGGCCAAGGGACCAAGCTGGAGATCAAA SEQ ID NO: 15 NI-302.2A2-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTACCTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAACCAGATGGAAGTGACAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGACGGCAGTGGCTGGAACGTCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 17 NI-302.2A2-V_(K)GACATCCAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTCTTTTATACACCTCCAAAAATAAGGACAGTAAGAACTACTTAGGTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAGTATTATACTACTCCTCAGTTCGGCGGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 19 NI-302.15D3-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTTCAGCCTGGGGGGTCCCTAAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGCACTGGGTCCGCCAAGCTCCAGGGAAGGGTCTGGTGTGGGTCTCACGTATTAGTAATGATGGCAGTAGCAAAACCTACGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAAAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAATACTTGGCGGATATTGTAGTAGTACCAGTTGTCGTCCCTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 135NI-302.15D3-V_(L)CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGTTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATTTTTGATGTCAGTAATCGGCCCTCAGGGATTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCGACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCATCCTA SEQ ID NO: 137 NI-302.64E5-V_(H)GAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCGACCAGGCCTGGATGAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGGATTAAAACGAAAACTGAGGGTGAAGCAACAGACTACGCAGCGCCCGTGAGAGGCAGATTCACCATCTCAAGAGATGATTCAGAAGACACGGTGTTTCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCCTGTATTACTGTACGTCAACGGGAGTCTTAGCAGCAGCTGTCGATGTCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 164NI-302.64E5-V_(K)GACATCCAGTTGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATGACCTGCAAGTCCAGCCAGAGTCTTTTCTACAGTTACAACAATGAGAACTACTTAGCCTGGTATCAGCAGAGACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTCAGACGTTCGGCCAAGGGACCAAAGTGGATATCAAA SEQ ID NO: 166

TABLE IV Nucleotide sequences of the V_(H) and V_(L) regionof antibodies recognizing an epitope of theC-terminal region of HTT, i.e exon 1 in aggregated form.Nucleotide sequences of variable heavy (V_(H)) and Antibodyvariable light (V_(L)) chains NI-302.35C1-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAAACTT GGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTACTGCCTCTGGATTCACCTTTAGTATAACG GCCCTGAGTTGGGTCCGCCAGGCTCCAGAAAAGGGGCCGCAGTGGGTCTCAGCAATCACTGGAA ATGCTTATGGGACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATTTCCAGAGACAACGC CAAGAACACACTGTACTTGCAAATGAACGGCCTGAGAGCCGAGGACACGGCCATCTATTACTGT GTGAAAGGAATTGCCTCCGATAGTAGTGGTTATTCTGCCTTCTGGGGCCCGGGCACCCTGGTCA CCGTCTCCTCG SEQ ID NO: 9NI-302.35C1-V_(K) GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT CCTGCAGGGCCAGTCAAAGTGTTGACAACCAGTTTGCCTGGTACCAACAGAAACCTGGCCAGGC TCCCAGGCTCCTCATTTATGATGCATCCAGGAGGGCCCCTGGCATCCCAGACAGGTTCAGTGGC AGTGGGTCTGGGACAGACTTCACTCTCACCATTAGCAGCCTAGAGCCTGAAGATTTCGCAATTT ATTACTGTCAGCATCGTTACACCTGGCTCTACACTTTTGGCCAGGGGACACGACTGGAGATTAA A SEQ ID NO: 11 NI-302.72F10-V_(H)GAGGTGCAGCTGGTGGAGACTGGGGGAGGCTT CGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCGGCAGTTAT GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTGTCAGATATCAGTGGTA TTGGTAGTAACACATACTACGCAGACTCCGTGAAGGGCCGTTTCACCATTTCCAGAGACAATTC CGACAATACGTTGTACCTGGACATGAGCAGCCTGAGAGCCGAGGACACGGCCAGATATTACTGT GCGAAGGATCGAAAGCGCAGTGGCTGGTACGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCT CCTCG SEQ ID NO: 176 NI-302.72F10-V_(K)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTT CGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCGGCAGTTAT GCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTGTCAGATATCAGTGGTA TTGGTAGTAACACATACTACGCAGACTCCGTGAAGGGCCGTTTCACCATTTCCAGAGACAATTC CGACAATACGTTGTACCTGGACATGAGCAGCCTGAGAGCCGAGGACACGGCCAGATATTACTGT GCGAAGGATCGAAAGCGCAGTGGCTGGTACGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCT CCTCG SEQ ID NO: 178

TABLE V Nucleotide sequences of the V_(H) and V_(L)region of antibodies recognizing HTT species and/or fragments thereof.Nucleotide sequences of variableheavy (V_(H)) and Antibodyvariable light (V_(L)) chains NI-302.6N9-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGT GCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGTCTCTGGATTCACCTTTAGTAGTTATGCCATGACC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGCCTGGGTCTCAACAATTAGTGCTACTGGTGGTAGTACAT TCTACACAGACTCCGTGAGGGGCCGGTTCACCATCTCCCGAGACAATTCCAAGAACACACTGTATCTGCA AATGAATAGCCTGAGAACCGACGACACGGCCATATATTATTGTGTGAAAGATCTATTTGGAGTGGACACC TCCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 21 NI-302.6N9-V_(K)GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTC TTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGCCCAGTCAGAGTGTCAGCGGCAGGTATGTGGCC TGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCTTCTATGCTGCATCCAACAGGGCCATTGGCA TCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGA AGATTTTGCAGTGTATTACTGTCAGCACTATGGTGCCTCATCGTACACTTTTGGCCCGGGGACCAAAGTG GATATCAAA SEQ ID NO: 23NI-302.8F1-V_(H) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGAAGCCGGGGGGGTCCCTTACAATCTCCTGTGCAG CCTCTGGTTTCACCTTCAGTAATGCCTGGATGAACTGGGTCCGCCAGGCTCCAGGTAAGGGGCTGGAGTG GGTCGGCCATATTAGAACGCAAGCTGAAGGAGGGACATCAGACTATGCTGCACCCGTGAAAGGCAGATTC ACCATCTCAAGAGATGACTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAG CCGTATATTATTGTATCCCCCCCCCCTACTACTACTATTACGGTCTGGACGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCCTCG SEQ ID NO: 81NI-302.8F1-V_(L) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTG GAGCCAGCAGTGATGTTGGGACTTATGACCTTGTCTCCTGGTACCAACAACATCCAGGCAAAGCCCCCAA ACTCATTATTTATGAGGTCAATAAGCGGCCCTCAGGGGTTTCTTATCGCTTCTCTGCCTCCAAGTCTGCC AACACGGCCTCCCTGACAATATCTGGGCTCCAGGCTGAGGACGAGGCTGAATATTACTGCTGCTCATATG CAGGTTATAGCACGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 83 NI-302.4A6-V_(H)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGCTTATGCCATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGTAGTGGTGGTAGTACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCTCCATCTCCAGAGACAACTCCAAAAACACCCTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTTCTGTGCGAAAGTTACCACGGAACTCTACGGT GCTAACTCCTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 184 NI-302.4A6-V_(K)GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTC TTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGTCAGCAGGTATTTAGCC TGGTACCAGCAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCA TCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGA AGATTTTGCAATGTATTACTGTCAGCTGTATGGTAACTCACAGACGTTCGGCCAGGGGACCAAGGTGGAG ATCAAA SEQ ID NO: 186NI-302.12H2-V_(H) GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTTTCCTGTGAAG CCTCTGGATTCACCTTTAGCAACTATGCCATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGTAATTAGTGGTACTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATC TCCAGAGACAATTCCATGAACACGCTGTATCTGCAAATGAACAGCCCGAGAGCCGACGACACGGCCGTAT ATTACTGTGCGAAAGATCTGAGGAAGATTAGCGGTCCTTTATACTACTACGGTATGGACGTCTGGGGCCA AGGGACCACGGTCACCGTCTCCTCGSEQ ID NO: 188 NI-302.12H2-V_(K) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTTTCCTGTGAAG CCTCTGGATTCACCTTTAGCAACTATGCCATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGTAATTAGTGGTACTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATC TCCAGAGACAATTCCATGAACACGCTGTATCTGCAAATGAACAGCCCGAGAGCCGACGACACGGCCGTAT ATTACTGTGCGAAAGATCTGAGGAAGATTAGCGGTCCTTTATACTACTACGGTATGGACGTCTGGGGCCA AGGGACCACGGTCACCGTCTCCTCGSEQ ID NO: 192 NI-302.8M1-V_(H) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAAGTTTCCTGCAAGG CATCCGGATACACCTTCACCATCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTG GATGGGAGGAATCAGCCCGAGTGGTGCCCACACAATGTACGCACAGAATTTCCAGGGCAGAGTCACCGTG ACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCGAGAGGGAGCACGGTGACTAACTATCGACCCTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCCTCG SEQ ID NO: 194NI-302.8M1-V_(K) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACTATCACTTGCC GGGCGAGTCAGGACATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAACTCCT GATCTTTGCTGCATCCACTTTGCAATCAGGGGTCCCGTCTCGGTTCGGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAACTATAACAGTG GCCCTCCGCCTTTCGGCCCTGGGACCAAAGTGGATATCAAA SEQ ID NO: 198

TABLE VI Nucleotide sequences of the V_(H) and V_(L) regionof antibodies an epitope of the N-terminl-region of HTT, i.e exon 1 in aggregated form Nucleotide sequences ofvariable heavy (V_(H)) and Antibody variable light (V_(L)) chainsNI-302.15E8-V_(H) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGC AGTCTCTGGATTCACCGTCAGTAGTTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCTCATACACTAGTAGTAGCAGAAGTAATACCAAAAAGTACGCAGACTCTGTGAAGGGCCGA TTCACCATCTCTAGAGACAATGCCAGGAACTCACTCTATCTGCAAATGAACAGCCTGAGAGACGAGGA CACGGCTGTGTATTACTGTGCGAGAGCAGGGGACTTCGGGGAGTTACTCACTGGTGAGGGGTATTACG GTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 131 NI-302.15E8-V_(L)TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCG TGTCCCCAGGACAGACAGCCACCATCACCTGCTCGGGAGATGAATTGGGGGATAAATATGTTGGTTGG TATCAACAGAAGCCAGGCCAGTCCCCTCTGCTGGTCATCTATCAAGATGCGAAGCGGCCCTCAGGGAT CCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTA TGGATGAGGCTGACTACTACTGTCAGGCGTGGGACAGCGGCACGATGGTTTTCGGCGGAGGGACCAGG CTGACCGTCCTA SEQ ID NO: 133

TABLE VII Nucleotide sequences of the V_(H) and V_(L) regionof antibodies recognizing an epitope of theQ/P-rich region of HTT, i.e exon 1 in aggregated form.Nucleotide sequences of variable heavy (V_(H)) and Antibodyvariable light (V_(L)) chains NI-302.7D8-V_(H)CAGGTGCAGCTGGTGCAATCTGGATCTGAGTTGA AGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACAACTTCAATAACTATGCCATC AATTGGTTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACACCATCACTGGGCA CCCAACGTATGCCCAGGGCTTCAAAGGACGATTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCAT ATCTGCAGATCAGCAGCCTAAAGCCTGAGGACACTGCCGTCTATTACTGTGCGAGAACTTACAGTAAC TACGGCGAATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 172 NI-302.7D8-V_(L)CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTG GGTCTCGTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGAAGTTATAACCTT GTCTCCTGGTACCAACAGTACCCAGGCAAGGCCCCCAAGCTCATAATTCATGAGGGCAGTGAGCGGCC CTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATTTCTGGGC TCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCTCATATGCAGGTACTACTACTTTCGTGCTATTC GGCGGAGGGACCAAGCTGACCGTCCTCSEQ ID NO: 174

Due to the cloning strategy the amino acid sequence at the N- andC-terminus of the heavy chain and light chains may potentially containprimer-induced alterations in FR1 and FR4, which however do notsubstantially affect the biological activity of the antibody. In orderto provide a consensus human antibody, the nucleotide and amino acidsequences of the original clone can be aligned with and tuned inaccordance with the pertinent human germ line variable region sequencesin the database; see, e.g., Vbase2, as described above. The amino acidsequence of human antibodies are indicated in bold when N- andC-terminus amino acids are considered to potentially deviate from theconsensus germ line sequence due to the PCR primer and thus have beenreplaced by primer-induced mutation correction (PIMC), see Table VI.Accordingly, in one embodiment of the present invention, thepolynucleotide comprises, consists essentially of, or consists of anucleic acid having a polynucleotide sequence of the VH and the VLregion of an anti-HTT antibody and/or fragments thereof as depicted inTable VI.

TABLE VIIINucleotide sequences of the V_(H) and V_(L) region of antibodies recognizing HTTspecies and/or fragments thereof showing replacement by PIMC (bold).Alternative Antibody-regions Nucleotide sequences of variable heavywith PIMC (VH) and variable light (VL) chains NI-302.33C11-PIMCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTTGTCCAGCCTGGGAACTCCCTGAGACTCTC V_(H)CTGTGCAGCGTCTGGATTCAGGTTCAGTGACTTTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGCTGGCACTTATATGGTATGATGGAGGGTATAAGTACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGATGTTTCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGACCCACCTAGAATATTGCAGTAGAACCACCTGCTATCTCGGCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCGSEQ ID NO: 97 NI-302.33C11-PIMCGACATCCAGTTGACCCAGTCTCCGTCCTTCCTATCTGCGTCTGTGGGAGACACAGTCACCTT V_(K)CACTTGCCGGGCCAGTCAGGGCATTAGCGATTATTTAGCCTGGTTTCAGCAGAAACCAGGGATTGCCCCTAAGCTCCTGATCTATGCTGCGTCCACTTTGCAAACCGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCCGCAGCCTGCAGTCTGAAGATTTTGGAACTTATTACTGTCAGCAGCTTAAAACTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 99 NI-302.63F3-PIMCGATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCAT V_(K)CAACTGCAAGTCCAATCAGAGTCTTTTCTACAGTTCCAACAATAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 101 NI-302.63F3-PIMC-GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCAT V_(K)CAACTGCAAGTCCTCACAGAGTCTTTTCTACAGTTCCAACAATAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 103 NI-302.63F3-PIMC-GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCAT V_(K)CAACTGCAAGTCCAATCAGGGCCTTTTCTACAGTTCCAACAATAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 105 NI-302.63F3-PIMC-GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCAT V_(K)CAACTGCAAGTCCCAACAGAGTCTTTTCTACAGTTCCAACAATAACAACTACTTAGCTTGGTACCAGCACAAATCCGGACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATCCGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTTCACTCTCACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATTACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 107 NI-302.35C1-PIMCGAAATTGTGCTGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCT V_(K)CTCCTGCAGGGCCAGTCAAAGTGTTGACAACCAGTTTGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATTTATGATGCATCCAGGAGGGCCCCTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATTAGCAGCCTAGAGCCTGAAGATTTCGCAATTTATTACTGTCAGCATCGTTACACCTGGCTCTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 109 NI-302.31F11-PIMCGATATTGTGATGACTCAATCACCACTCTCCCTGCCCGTCGCCCCTGGAGAGCCGGCCTCCAT V_(K)CTCCTGCAGGTCTAGTCAGAGCCTCCTATACAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGAAGCCTCCACAGCTCCTGGTCTATTTGGGTTCTGATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCAAAGATTTTACACTGAACATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 111 NI-302.2A2-PIMCGATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCAT V_(K)CAACTGCAAGTCCAGCCAGAGTCTTTTATACACCTCCAAAAATAAGGACAGTAAGAACTACTTAGGTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAGTATTATACTACTCCTCAGTTCGGCGGAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 113 NI-302.74C11-PIMCCAGGTGCAGCTGGTGCAATCTGGGACTGAGGTGCAGAAGCCTGGGGCCTCAGTAAAAGTCTC V_(H)CTGCAAGGCTTCTGGATACAGTTTCACCGGCTACTTTTTGCACTGGGTACGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGTGGATCAACCCTAACAGTGGTGACACAAACTATGCAGAGAAGTTTCGGGGCAGAATCATCATGACCAGGGACACGTCTGTCAGCACAGCCCACATGGAGTTGAGCAGCCTGAGATTTGACGACACGGCCCTATATTACTGTACGAGAGAGGCCCCTGACCCGGGCGCTGAGACGGACGTCTGGGGCCAAGGAACCACGGTCACCGTCTCCTCG SEQ ID NO: 115NI-302.74C11-PIMCTCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCAC V_(L)CTGCTCTGGAGATGCAGTGCCAAAGCAGTATATTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTATTCTGGTGATATATAAAGACACTCAGAGGCCTTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCAGGGACAACAGTCACGTTGACCATAACTGGCGTCCAGGCAGACGACGAGGGTGACTATTACTGTCAATCAGCAGACAGTAGTGCTACTTGGGTGTTCGGCGGAGGGACCAAATTGACCGTCCTA SEQ ID NO: 117 NI-302.39G12-PIMCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCACCCTTGGGGGTCCCTGAGACTCTC V_(H)CTGTGCAGCCTCTGGATTCAGCGTCTCTAATTACGCCATAACTTGGGTCCGCCGGGCTCCAGGGAAGGGGCTGCAATATATTTCAGTAATTTATCGTGATGGCAGGACATACTACGGAGACTCCGTGAGGGGCCGCTTCACCATCTCTAGGGACGATTCCAAGAACACTCTCTATCTTCAAATGAACAGCCTGAGATTTGAGGACACGGCTGTGTATTACTGTGCGAGAGCGCACGGCCAATATTACTATGGTGTGGACGTCTGGGGCCAAGGAACCACGGTCACCGTCTCCTCG SEQ ID NO: 119NI-302.39G12-PIMCGACATCGTGATGACCCAGTCTCCACTCTCCCTGTCCGTCAGCCCTGGAGAGCCGGCCTCCAT V_(K)CTCCTGCAGGTCTAGTCAGAGCCTCCTACATAGTAATGGATACAACTATTTGGATTGGTACCGGCAGAAACCAGGGCAGTCTCCACAGCTCCTGATCTATTTGAGTTCTAATCGGCCCTCCGGGGTCCCTGATAGGTTCAGTGCCAGTGGATCAGGCACAGAGTTCACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 121 NI-302.11 A4-PIMCGAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGAGAAAGAGCCACCCT V_(K)CTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTCGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTACGTCCCGCAGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAACAGTATGGTAGCTCGTGGACGTTCGGCCCAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 123 NI-302.22H9-PIMCGATATTGTGATGACTCAATCACCACTCTCCCTGTCCGTCAGCCCTGGAGAGCCGGCCTCCAT V_(K)CTCCTGCAGGTCTAGTCAGAGCCTCCTACATAGTAATGGATACAACTATTTGGATTGGTACCGGCAGAAACCAGGGCAGTCTCCACAACTCCTGATCTATTTGAATTCTAATCGGGCCTCCGGGGTCCCTGATAGGTTCAGTGGCAGTGGATCAGGCACAGAGTTCACACTGACAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 125 NI-302.44D7-PIMCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTC V_(H)CTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTATAGTGATACTAGCACATATTACGCAGACTCCGTGAAGGGCCGCTTCACCGTCTCCAGAGACATTTCCAAGAACACGCTGTATCTGCAAATGAATAGCCTGAGGGCCGAGGACACGGCCGTATATTACTGCGCGAAAGGTACCAGGGACTATTACGGTATGGACGTCTGGGGCCAAGGAACCACGGTCACCGTCTCCTCG SEQ ID NO: 127NI-302.78H12-PIMCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC V_(H)CTGTCTTGTCTCTAGTTACTCCATCAGCAATGGTTACTACTGGGGCTGGATTCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATCATAATGGGAACACCTATTACAACCCGTCCCTCAAGAGTCGAGTCATCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGTTGAGGTCTGTGACCGCCGCAGACACGGCCGTGTACTACTGTGCGATGCCAAGTGCCACCTATTATTATGGTTCGGGGACTCAATTCCATGCGTTTGATGTCTGGGGCCAAGGGACAATGGTCACC GTCTCTTCGSEQ ID NO: 129 NI-302.64E5-PIMCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTC V_(H)CTGTGCAGCCTCTGGATTCACTTTCGACCAGGCCTGGATGAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGGATTAAAACGAAAACTGAGGGTGAAGCAACAGACTACGCAGCGCCCGTGAGAGGCAGATTCACCATCTCAAGAGATGATTCAGAAGACACGGTGTTTCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCCTGTATTACTGTACGTCAACGGGAGTCTTAGCAGCAGCTGTCGATGTCTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 166NI-302.64E5-PIMCGATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCAT V_(K)GACCTGCAAGTCCAGCCAGAGTCTTTTCTACAGTTACAACAATGAGAACTACTTAGCCTGGTATCAGCAGAGACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTCAGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 170 NI-302.72F10-PIMCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTCGTACAGCCTGGGGGGTCCCTGAGACTCTC V_(H)CTGTGCAGCCTCTGGATTCAACTTCGGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTGTCAGATATCAGTGGTATTGGTAGTAACACATACTACGCAGACTCCGTGAAGGGCCGTTTCACCATTTCCAGAGACAATTCCGACAATACGTTGTACCTGGACATGAGCAGCCTGAGAGCCGAGGACACGGCCAGATATTACTGTGCGAAGGATCGAAAGCGCAGTGGCTGGTACGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 178NI-302.72F10-PIMCGAAATTGTGCTGACTCAGTCTCCAGCCACCCTGACTTTGTCTCCAGGGGAAAGAGCCACCCT V_(K)CTCCTGCAGGGCCAGTCAGAGTATTAGCGCCTACTTAGGCTGGTATCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCATTAGGGCCACTGGCATTCCAGACAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTCTGCAGTTTATTACTGTCACCAGCGTAGCAAGTGGCCTCTTACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 182 NI-302.12H2-PIMCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTTTC V_(H)CTGTGAAGCCTCTGGATTCACCTTTAGCAACTATGCCATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTAATTAGTGGTACTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCATGAACACGCTGTATCTGCAAATGAACAGCCCGAGAGCCGACGACACGGCCGTATATTACTGTGCGAAAGATCTGAGGAAGATTAGCGGTCCTTTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCGSEQ ID NO: 190 NI-302.8M1-PIMCCAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAAGTTTC V_(H)CTGCAAGGCATCCGGATACACCTTCACCATCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGAATCAGCCCGAGTGGTGCCCACACAATGTACGCACAGAATTTCCAGGGCAGAGTCACCGTGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGAGCACGGTGACTAACTATCGACCCTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 196

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotideswhich encode fusion polynucleotides, Fab fragments, and otherderivatives, as described herein, are also contemplated by theinvention. The polynucleotides may be produced or manufactured by anymethod known in the art. For example, if the nucleotide sequence of theantibody is known, a polynucleotide encoding the antibody may beassembled from chemically synthesized oligonucleotides, e.g., asdescribed in Kutmeier et al., BioTechniques 17 (1994), 242, which,briefly, involves the synthesis of overlapping oligonucleotidescontaining portions of the sequence encoding the antibody, annealing andligating of those oligonucleotides, and then amplification of theligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody, or antigen-bindingfragment, variant, or derivative thereof may be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA⁺RNA, isolated from, any tissue or cellsexpressing the HTT-specific antibody, such as hybridoma cells selectedto express an antibody) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence may be manipulated usingmethods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

IV. Expression of Antibody Polypeptides

Following manipulation of the isolated genetic material to provideantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, the polynucleotides encoding the antibodiesare typically inserted in an expression vector for introduction intohost cells that may be used to produce the desired quantity of antibody.Recombinant expression of an antibody, or fragment, derivative, oranalog thereof, e.g., a heavy or light chain of an antibody which bindsto a target molecule is described herein. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablelinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,international applications WO 86/05807 and WO 89/01036; and U.S. Pat.No. 5,122,464) and the variable domain of the antibody may be clonedinto such a vector for expression of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses, and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells. For the purposes of this invention, numerousexpression vector systems may be employed. For example, one class ofvector utilizes DNA elements which are derived from animal viruses suchas bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus,baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus. Othersinvolve the use of polycistronic systems with internal ribosome bindingsites. Additionally, cells which have integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow selection of transfected host cells. The marker may provide forprototrophy to an auxotrophic host, biocide resistance (e.g.,antibiotics), or resistance to heavy metals such as copper. Theselectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include signal sequences, splicesignals, as well as transcriptional promoters, enhancers, andtermination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) as discussed above. Inone embodiment, this is accomplished using a proprietary expressionvector of Biogen IDEC, Inc., referred to as NEOSPLA, and disclosed inU.S. Pat. No. 6,159,730. This vector contains the cytomegaloviruspromoter/enhancer, the mouse beta globin major promoter, the SV40 originof replication, the bovine growth hormone polyadenylation sequence,neomycin phosphotransferase exon 1 and exon 2, the dihydrofolatereductase gene, and leader sequence. This vector has been found toresult in very high level expression of antibodies upon incorporation ofvariable and constant region genes, transfection in CHO cells, followedby selection in G418 containing medium and methotrexate amplification.Of course, any expression vector which is capable of elicitingexpression in eukaryotic cells may be used in the present invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those which express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation which can be carried out, forexample, by robotic systems. Vector systems are also taught in U.S. Pat.Nos. 5,736,137 and 5,658,570, each of which is incorporated by referencein its entirety herein. This system provides for high expression levels,e.g., >30 pg/cell/day. Other exemplary vector systems are disclosede.g., in U.S. Pat. No. 6,413,777.

In other preferred embodiments the antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may beexpressed using polycistronic constructs such as those disclosed in USpatent application publication no. 2003-0157641 A1 and incorporatedherein in its entirety. In these expression systems, multiple geneproducts of interest such as heavy and light chains of antibodies may beproduced from a single polycistronic construct. These systemsadvantageously use an internal ribosome entry site (IRES) to providerelatively high levels of antibodies. Compatible IRES sequences aredisclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein.Those skilled in the art will appreciate that such expression systemsmay be used to effectively produce the full range of antibodiesdisclosed in the instant application. Therefore, in one embodiment thepresent invention provides a vector comprising the polynucleotideencoding at least the binding domain or variable region of animmunoglobulin chain of the antibody, optionally in combination with apolynucleotide that encodes the variable region of the otherimmunoglobulin chain of said binding molecule.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the antibody has been prepared, the expression vector may beintroduced into an appropriate host cell. Introduction of the plasmidinto the host cell can be accomplished by various techniques well knownto those of skill in the art. These include, but are not limited to,transfection including lipotransfection using, e.g., Fugene® orlipofectamine, protoplast fusion, calcium phosphate precipitation, cellfusion with enveloped DNA, microinjection, and infection with intactvirus. Typically, plasmid introduction into the host is via standardcalcium phosphate co-precipitation method. The host cells harboring theexpression construct are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells comprising apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, or at least the binding domain or variable regionof an immunoglobulin thereof, which preferably are operable linked to aheterologous promoter. In addition or alternatively the invention alsoincludes host cells comprising a vector, as defined hereinabove,comprising a polynucleotide encoding at least the binding domain orvariable region of an immunoglobulin chain of the antibody, optionallyin combination with a polynucleotide that encodes the variable region ofthe other immunoglobulin chain of said binding molecule. In preferredembodiments for the expression of double-chained antibodies, a singlevector or vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad.Sci. USA 77 (1980), 2197. The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., Escherichia coli, Bacillussubtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,NSO, BLK, 293, 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,bacterial cells such as E. coli, and more preferably, eukaryotic cells,especially for the expression of whole recombinant antibody molecule,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese Hamster Ovary (CHO) cells, inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies; see, e.g., Foecking et al., Gene 45 (1986), 101;Cockett et al., Bio/Technology 8 (1990), 2.

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO (Chinese HamsterOvary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA(human cervical carcinoma), CVI (monkey kidney line), COS (a derivativeof CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK,WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast),HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). CHO and 293 cells are particularly preferred.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalskaand Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adeninephosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl.Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78(1981), 2072); neo, which confers resistance to the aminoglycoside G-418Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu,Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32(1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan andAnderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993),155-215; and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30 (1984), 147. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, N Y (1990); and in Chapters 12 and 13, Dracopoli et al.(eds), Current Protocols in Human Genetics, John Wiley & Sons, N Y(1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification, for a review; see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase; see Crouse et al., Mol. Cell. Biol. 3(1983), 257.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-) affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies, or antigen-binding fragments, variants orderivatives thereof of the invention can also be expressed innon-mammalian cells such as bacteria or insect or yeast or plant cells.Bacteria which readily take up nucleic acids include members of theenterobacteriaceae, such as strains of E. coli or Salmonella;Bacillaceae, such as B. subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies; see, e.g., internationalapplication WO 02/096948.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983),1791), in which the antibody coding sequence may be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res.13 (1985), 3101-3109; Van Heeke and Schuster, J. Biol. Chem. 24 (1989),5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrix ofglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al.,Gene 10 (1980), 157) is commonly used. This plasmid already contains theTRP1 gene which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example ATCC No. 44076 orPEP4-1 (Jones, Genetics 85 (1977), 12). The presence of the trp1 lesionas a characteristic of the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingfor example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,e.g. ammonium sulfate precipitation, or by any other standard techniquefor the purification of proteins; see, e.g., Scopes, “ProteinPurification”, Springer Verlag, N.Y. (1982). Alternatively, a preferredmethod for increasing the affinity of antibodies of the invention isdisclosed in US patent publication 2002-0123057 A1. In one embodimenttherefore, the present invention also provides a method for preparing ananti-HTT antibody or an antibody recognizing mutated and/or aggregatedHTT species and/or fragments thereof or immunoglobulin chain(s) thereof,said method comprising:

-   (a) culturing the host cell as defined hereinabove, which cell    comprised a polynucleotide or a vector as defined hereinbefore; and-   (b) isolating said antibody or immunoglobulin chain(s) thereof from    the culture.

Furthermore, in one embodiment the present invention also relates to anantibody or immunoglobulin chain(s) thereof encoded by a polynucleotideas defined hereinabove or obtainable by said method for preparing ananti-HTT antibody or an antibody recognizing mutated and/or aggregatedHTT species and/or fragments thereof or immunoglobulin chain(s) thereof.

V. Fusion Proteins and Conjugates

In certain embodiments, the antibody polypeptide comprises an amino acidsequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain Fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label such as afluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and thelike)

An antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulinHTT-binding domain with at least one target binding site, and at leastone heterologous portion, i.e., a portion with which it is not naturallylinked in nature. The amino acid sequences may normally exist inseparate proteins that are brought together in the fusion polypeptide orthey may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an antibody, or an antigen-binding fragment, variant, oranalog thereof is derived from a non-immunoglobulin polypeptide of thesame species, or an immunoglobulin or non-immunoglobulin polypeptide ofa different species.

As discussed in more detail elsewhere herein, antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, antibodies may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins; see, e.g., international applications WO92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; andEuropean patent application EP 0 396 387.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids.

Antibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theantibody, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini, or on moieties such as carbohydrates. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given antibody. Also,a given antibody may contain many types of modifications. Antibodies maybe branched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicantibodies may result from posttranslational natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, e.g., Proteins—Structure And Molecular Properties, T. E. Creighton,W. H. Freeman and Company, New York 2nd Ed., (1993); PosttranslationalCovalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press,New York, (1983) 1-12; Seifter et al., Meth. Enzymol. 182 (1990),626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The present invention also provides for fusion proteins comprising anantibody, or antigen-binding fragment, variant, or derivative thereof,and a heterologous polypeptide. In one embodiment, a fusion protein ofthe invention comprises, consists essentially of, or consists of, apolypeptide having the amino acid sequence of any one or more of theV_(H) regions of an antibody of the invention or the amino acid sequenceof any one or more of the V_(L) regions of an antibody of the inventionor fragments or variants thereof, and a heterologous polypeptidesequence. In another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises, consistsessentially of, or consists of a polypeptide having the amino acidsequence of any one, two, three of the V_(H)-CDRs of an antibody, orfragments, variants, or derivatives thereof, or the amino acid sequenceof any one, two, three of the V_(L)-CDRs of an antibody, or fragments,variants, or derivatives thereof, and a heterologous polypeptidesequence. In one embodiment, the fusion protein comprises a polypeptidehaving the amino acid sequence of a V_(H)-CDR3 of an antibody of thepresent invention, or fragment, derivative, or variant thereof, and aheterologous polypeptide sequence, which fusion protein specificallybinds to HTT. In another embodiment, a fusion protein comprises apolypeptide having the amino acid sequence of at least one V_(H) regionof an antibody of the invention and the amino acid sequence of at leastone V_(L) region of an antibody of the invention or fragments,derivatives or variants thereof, and a heterologous polypeptidesequence. Preferably, the V_(H) and V_(L) regions of the fusion proteincorrespond to a single source antibody (or scFv or Fab fragment) whichspecifically binds HTT. In yet another embodiment, a fusion protein foruse in the diagnostic and treatment methods disclosed herein comprises apolypeptide having the amino acid sequence of any one, two, three, ormore of the V_(H) CDRs of an antibody and the amino acid sequence of anyone, two, three, or more of the V_(L) CDRs of an antibody, or fragmentsor variants thereof, and a heterologous polypeptide sequence.Preferably, two, three, four, five, six, or more of the V_(H)-CDR(s) orV_(L)-CDR(s) correspond to single source antibody (or scFv or Fabfragment) of the invention. Nucleic acid molecules encoding these fusionproteins are also encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84(1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531;Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA CellBiol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990),667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167);CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley etal., J. Exp. Med. 173 (1991), 721-730); CTLA-4 (Lisley et al., J. Exp.Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991),1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991),2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991);and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991),Abstract No. 1448).

As discussed elsewhere herein, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be fused toheterologous polypeptides to increase the in vivo half-life of thepolypeptides or for use in immunoassays using methods known in the art.For example, in one embodiment, PEG can be conjugated to the antibodiesof the invention to increase their half-life in vivo; see, e.g., Leonget al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002),531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.Moreover, antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide (HIS), such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37 (1984),767), GST, c-mycand the “flag” tag; see, e.g., Bill Brizzard,BioTechniques 44 (2008) 693-695 for a review of epitope taggingtechniques, and Table 1 on page 694 therein listing the most commonepitope tags usable in the present invention, the subject matter ofwhich is hereby expressly incorporated by reference.

Fusion proteins can be prepared using methods that are well known in theart; see for example U.S. Pat. Nos. 5,116,964 and 5,225,538. The precisesite at which the fusion is made may be selected empirically to optimizethe secretion or binding characteristics of the fusion protein. DNAencoding the fusion protein is then transfected into a host cell forexpression, which is performed as described hereinbefore.

Antibodies of the present invention may be used in non-conjugated formor may be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed. In particular, antibodies,or antigen-binding fragments, variants, or derivatives thereof of theinvention may be conjugated to therapeutic agents, prodrugs, peptides,proteins, enzymes, viruses, lipids, biological response modifiers,pharmaceutical agents, or PEG.

Conjugates that are immunotoxins including conventional antibodies havebeen widely described in the art. The toxins may be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The antibodies of the present invention can be used in a correspondingway to obtain such immunotoxins. Illustrative of such immunotoxins arethose described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and byFanger, Immunol. Today 12 (1991), 51-54.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared,e.g., by reacting a HTT-binding polypeptide with an activated ester ofbiotin such as the biotin N-hydroxysuccinimide ester. Similarly,conjugates with a fluorescent marker may be prepared in the presence ofa coupling agent, e.g. those listed herein, or by reaction with anisothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of theantibodies, or antigen-binding fragments, variants or derivativesthereof of the invention are prepared in an analogous manner.

The present invention further encompasses antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention conjugatedto a diagnostic or therapeutic agent. The antibodies can be useddiagnostically to, for example, demonstrate presence of a HTTamyloidosis to indicate the risk of getting a disease or disorderassociated with mutated and/or aggregated HTT, to monitor thedevelopment or progression of such a disease, i.e. a disease showing theoccurrence of, or related to aggregated HTT, or as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatmentand/or prevention regimen. In one embodiment thus, the present inventionrelates to an antibody, which is detectably labeled. Furthermore, in oneembodiment, the present invention relates to an antibody, which isattached to a drug. Detection can be facilitated by coupling theantibody, or antigen-binding fragment, variant, or derivative thereof toa detectable substance. The detectable substances or label may be ingeneral an enzyme; a heavy metal, preferably gold; a dye, preferably afluorescent or luminescent dye; or a radioactive label. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions; see,e.g., U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as diagnostics according to the present invention.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹Tc. Therefore, in one embodiment the present invention provides adetectably labeled antibody, wherein the detectable label is selectedfrom the group consisting of an enzyme, a radioisotope, a fluorophoreand a heavy metal.

An antibody, or antigen-binding fragment, variant, or derivative thereofalso can be detectably labeled by coupling it to a chemiluminescentcompound. The presence of the chemiluminescent-tagged antibody is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol.31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio,(ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980);Ishikawa, et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981).The enzyme, which is bound to the antibody, will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibody, orantigen-binding fragment, variant, or derivative thereof, it is possibleto detect the antibody through the use of a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,(March, 1986)), which is incorporated by reference herein). Theradioactive isotope can be detected by means including, but not limitedto, a gamma counter, a scintillation counter, or autoradiography.

An antibody, or antigen-binding fragment, variant, or derivative thereofcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to an antibody, orantigen-binding fragment, variant, or derivative thereof are well known,see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), Marcel Dekker, Inc., (1987) 623-53;Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), (1985) 475-506; “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press (1985)303-16, and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. 62 (1982), 119-158.

As mentioned, in certain embodiments, a moiety that enhances thestability or efficacy of a binding molecule, e.g., a bindingpolypeptide, e.g., an antibody or immunospecific fragment thereof can beconjugated. For example, in one embodiment, PEG can be conjugated to thebinding molecules of the invention to increase their half-life in vivo.Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

VI. Compositions and Methods of Use

The present invention relates to compositions comprising theaforementioned HTT-binding molecule, e.g., antibody or antigen-bindingfragment thereof of the present invention or derivative or variantthereof, or the polynucleotide, vector or cell of the invention asdefined hereinbefore. In one embodiment, the composition of the presentinvention is a pharmaceutical composition and further comprises apharmaceutically acceptable carrier. Furthermore, the pharmaceuticalcomposition of the present invention may comprise further agents such asinterleukins or interferons depending on the intended use of thepharmaceutical composition. For use in the treatment of a disease ordisorder showing the occurrence of, or related to mutated and/oraggregated HTT, such as HTT amyloidosis, the additional agent may beselected from the group consisting of small organic molecules, anti-HTTantibodies, and combinations thereof. Hence, in a particular preferredembodiment the present invention relates to the use of the HTT-bindingmolecule, e.g., antibody or antigen-binding fragment thereof of thepresent invention or of a binding molecule having substantially the samebinding specificities of any one thereof, the polynucleotide, the vectoror the cell of the present invention for the preparation of apharmaceutical or diagnostic composition for prophylactic andtherapeutic treatment of Huntington's disease (HD) and/or a disease ordisorder associated with HTT and/or HTT amyloidosis, monitoring theprogression of HD and/or a disease or disorder associated with HTTand/or HTT amyloidosis or a response to a HTT amyloidosis treatment in asubject or for determining a subject's risk for developing a disease ordisorder associated with HTT.

Hence, in one embodiment the present invention relates to a method oftreating a disease or disorder characterized by abnormal accumulationand/or deposition of HTT and/or aggregated and/or mutated HTT inaffected systems and organs which method comprises administering to asubject in need thereof a therapeutically effective amount of any one ofthe afore-described HTT-binding molecules, antibodies, polynucleotides,vectors or cells of the instant invention.

A particular advantage of the therapeutic approach of the presentinvention lies in the fact that the recombinant antibodies of thepresent invention are derived from B cells or memory B cells fromhealthy human subjects with no signs or symptoms of a disease, e.g.carrying an asymptomatic mutation and/or mutations, showing theoccurrence of, or related to aggregated HTT and thus are, with a certainprobability, capable of preventing a clinically manifest disease relatedto mutated and/or aggregated HTT, or of diminishing the risk of theoccurrence of the clinically manifest disease or disorder, or ofdelaying the onset or progression of the clinically manifest disease ordisorder. Typically, the antibodies of the present invention also havealready successfully gone through somatic maturation, i.e. theoptimization with respect to selectivity and effectiveness in the highaffinity binding to the target HTT molecule by means of somaticvariation of the variable regions of the antibody.

The knowledge that such cells in vivo, e.g. in a human, have not beenactivated by means of related or other physiological proteins or cellstructures in the sense of an autoimmunological or allergic reaction isalso of great medical importance since this signifies a considerablyincreased chance of successfully living through the clinical testphases. So to speak, efficiency, acceptability and tolerability havealready been demonstrated before the preclinical and clinicaldevelopment of the prophylactic or therapeutic antibody in at least onehuman subject. It can thus be expected that the human-derived anti-HTTantibodies of the present invention, both its target structure-specificefficiency as therapeutic agent and its decreased probability of sideeffects significantly increase its clinical probability of success.

The present invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, e.g. anti-HTT antibody,binding fragment, derivative or variant thereof, polynucleotide, vectoror cell of the present invention. Associated with such container(s) canbe a notice in the form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition or alternatively the kit comprisesreagents and/or instructions for use in appropriate diagnostic assays.The composition, e.g. kit of the present invention is of courseparticularly suitable for the risk assessment, diagnosis, prevention andtreatment of Huntington's disease and/or a disease or disorder which isaccompanied with the presence of mutated and/or aggregated HTT, and inparticular applicable for the treatment of disorders generallycharacterized by HTT amyloidosis.

The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, intranasal,topical or intradermal administration or spinal or brain delivery.Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are preferably adjusted to a pHand isotonic state compatible with the nasal mucous membranes.Formulations for rectal or vaginal administration may be presented as asuppository with a suitable carrier.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, preferably atleast 1 mg/kg. Doses intermediate in the above ranges are also intendedto be within the scope of the invention. Subjects can be administeredsuch doses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimensentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline, and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases, and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as dopamine orpsychopharmacologic drugs, depending on the intended use of thepharmaceutical composition.

Furthermore, in a preferred embodiment of the present invention thepharmaceutical composition may be formulated as a vaccine, for example,if the pharmaceutical composition of the invention comprises an anti-HTTantibody or HTT-binding fragment, derivative or synthetic orbiotechnological variant thereof for passive immunization. As mentionedin the background section mutated and/or aggregated HTT species and/orfragments or derivatives thereof are a major trigger for HTTamyloidosis. Accordingly, it is prudent to expect that passiveimmunization with human anti-HTT antibodies and equivalent HTT-bindingmolecules of the present invention will help to circumvent severaladverse effects of active immunization therapy concepts and lead to areduced aggregation of HTT. Therefore, the present anti-HTT antibodiesand their equivalents of the present invention will be particularlyuseful as a vaccine for the prevention or amelioration of diseases ordisorders showing the presence of, or caused by aggregated HTT such asHD.

In one embodiment, it may be beneficial to use recombinant Fab (rFab)and single chain fragments (scFvs) of the antibody of the presentinvention, which might more readily penetrate a cell membrane. Forexample, Robert et al., Protein Eng. Des. Sel. (2008); 51741-0134,published online ahead, describe the use of chimeric recombinant Fab(rFab) and single chain fragments (scFvs) of monoclonal antibody WO-2which recognizes an epitope in the N-terminal region of Abeta. Theengineered fragments were able to (i) prevent amyloid fibrillization,(ii) disaggregate preformed Abeta1-42 fibrils and (iii) inhibitAbeta1-42 oligomer-mediated neurotoxicity in vitro as efficiently as thewhole IgG molecule. The perceived advantages of using small Fab and scFvengineered antibody formats which lack the effector function includemore efficient passage across the blood-brain barrier and minimizing therisk of triggering inflammatory side reactions. Furthermore, besidesscFv and single-domain antibodies retain the binding specificity offull-length antibodies, they can be expressed as single genes andintracellularly in mammalian cells as intrabodies, with the potentialfor alteration of the folding, interactions, modifications, orsubcellular localization of their targets; see for review, e.g., Millerand Messer, Molecular Therapy 12 (2005), 394-401.

In a different approach Muller et al., Expert Opin. Biol. Ther. (2005),237-241, describe a technology platform, so-called ‘SuperAntibodyTechnology’, which is said to enable antibodies to be shuttled intoliving cells without harming them. Such cell-penetrating antibodies opennew diagnostic and therapeutic windows. The term ‘TransMabs’ has beencoined for these antibodies.

In a further embodiment, co-administration or sequential administrationof other antibodies useful for treating a disease, disorder, or symptomsrelated to the occurrence of mutated and/or aggregated HTT may bedesirable. In one embodiment, the additional antibody is comprised inthe pharmaceutical composition of the present invention. Examples ofantibodies which can be used to treat a subject include, but are notlimited to, antibodies targeting CD33, SGLT2, IL-6, and IL-1.

In a further embodiment, co-administration or sequential administrationof other agents useful for treating a disease, disorder, or symptomsrelated to mutated and/or aggregated HTT, may be desirable. In oneembodiment, the additional agent is comprised in the pharmaceuticalcomposition of the present invention. Examples of agents which can beused to treat a subject include, but are not limited to: VMAT2inhibitors targeting involuntary muscle movements such as Xenazine™,anti-inflammatory agents such as diflusinal, corticosteroids,2-(2,6-dichloranilino) phenylacetic acid (diclofenac),iso-butyl-propanoic-phenolic acid (ibuprofen); diuretics,Epigallocatechin gallate, Melphalan hydrochloride, dexamethasone,Bortezomib, Bortezomib-Melphalan, Bortezomib-dexamethasone,Melphalan-dexamethasone, Bortezomib-Melphalan-dexamethasone;antidepressants, antipsychotic drugs, neuroleptics, antidementiva (e.g.the NMDA-rezeptor antagonist memantine), acetylcholinesterase inhibitors(e.g. Donepezil, HCl, Rivastigmine, Galantamine), glutamat-antagonistsand other nootropics blood pressure medication (e.g. Dihydralazin,Methyldopa), cytostatics, glucocorticoides,angiotensin-converting-enzyme (ACE) inhibitors; anti-inflammatory agentsor any combination thereof.

A therapeutically effective dose or amount refers to that amount of theactive ingredient sufficient to ameliorate the symptoms or condition.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀.

From the foregoing, it is evident that the present invention encompassesany use of an HTT-binding molecule and/or fragments thereof comprisingat least one CDR of the above described antibody, in particular fordiagnosing and/or treatment of a disease or disorder related to mutatedand/or aggregated HTT species and/or fragments thereof as mentionedabove, such as HD and/or HTT amyloidosis. Preferably, said bindingmolecule is an antibody of the present invention or an immunoglobulinchain thereof. In addition, the present invention relates toanti-idiotypic antibodies of any one of the mentioned antibodiesdescribed hereinbefore. These are antibodies or other binding moleculeswhich bind to the unique antigenic peptide sequence located on anantibody's variable region near the antigen-binding site and are useful,e.g., for the detection of anti-HTT antibodies in a sample obtained froma subject. In one embodiment thus, the present invention provides anantibody as defined hereinabove and below or a HTT-binding moleculehaving substantially the same binding specificities of any one thereof,the polynucleotide, the vector or the cell as defined herein or apharmaceutical or diagnostic composition comprising any one thereof foruse in prophylactic treatment, therapeutic treatment and/or monitoringthe progression or a response to treatment of a disease or disorderrelated to HTT, preferably wherein the disorder is associated with HTTamyloidosis, such as Huntington's disease (HD).

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described HTT-bindingmolecules, antibodies, antigen-binding fragments, polynucleotides,vectors or cells of the invention and optionally suitable means fordetection such as reagents conventionally used in immuno- or nucleicacid-based diagnostic methods. The antibodies of the invention are, forexample, suited for use in immunoassays in which they can be utilized inliquid phase or bound to a solid phase carrier. Examples of immunoassayswhich can utilize the antibody of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA), thesandwich (immunometric assay), flow cytometry, and the Western blotassay. The antigens and antibodies of the invention can be bound to manydifferent carriers and used to isolate cells specifically bound thereto.Examples of well-known carriers include glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amyloses, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble orinsoluble for the purposes of the invention. There are many differentlabels and methods of labeling known to those of ordinary skill in theart. Examples of the types of labels which can be used in the presentinvention include enzymes, radioisotopes, colloidal metals, fluorescentcompounds, chemiluminescent compounds, and bioluminescent compounds; seealso the embodiments discussed hereinabove.

By a further embodiment, the HTT-binding molecules, in particularantibodies of the present invention may also be used in a method for thediagnosis of a disease or disorder in an individual by obtaining a bodyfluid sample from the tested individual which may be a blood sample, aplasma sample, a serum sample, a lymph sample or any other body fluidsample, such as a saliva or a urine sample and contacting the body fluidsample with an antibody of the instant invention under conditionsenabling the formation of antibody-antigen complexes. The level of suchcomplexes is then determined by methods known in the art, a levelsignificantly higher than that formed in a control sample indicating thedisease or disorder in the tested individual. In the same manner, thespecific antigen bound by the antibodies of the invention may also beused. Thus, the present invention relates to an in vitro immunoassaycomprising the binding molecule, e.g., antibody or antigen-bindingfragment thereof of the invention.

In a further embodiment of the present invention the HTT-bindingmolecules, in particular antibodies of the present invention may also beused in a method for the diagnosis of a disease or disorder in anindividual by obtaining a biopsy from the tested individual.

In this context, the present invention also relates to meansspecifically designed for this purpose. For example, an antibody-basedarray may be used, which is for example loaded with antibodies orequivalent antigen-binding molecules of the present invention whichspecifically recognize HTT. Design of microarray immunoassays issummarized in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696.Accordingly, the present invention also relates to microarrays loadedwith HTT-binding molecules identified in accordance with the presentinvention.

In one embodiment, the present invention relates to a method ofdiagnosing a disease or disorder related to mutated and/or aggregatedHTT species and/or fragments thereof in a subject, the method comprisingdetermining the presence of HTT and/or mutated and/or aggregated HTT ina sample from the subject to be diagnosed with at least one antibody ofthe present invention, a HTT-binding fragment thereof or an HTT-bindingmolecule having substantially the same binding specificities of any onethereof, wherein the presence of pathologically mutated and/oraggregated HTT is indicative for HD and/or HTT amyloidosis and anincrease of the level of the pathologically mutated and/or aggregatedHTT in comparison to the level of the physiological HTT is indicativefor progression of HD and/or HTT amyloidosis in said subject.

The subject to be diagnosed may be asymptomatic or preclinical for thedisease. Preferably, the control subject has a disease associated withmutated and/or aggregated HTT, e.g. Huntington's disease (HD), wherein asimilarity between the level of pathologically mutated and/or aggregatedHTT and the reference standard indicates that the subject to bediagnosed has a HTT amyloidosis or is at risk to develop a HTTamyloidosis. Alternatively, or in addition as a second control thecontrol subject does not have a HTT amyloidosis, wherein a differencebetween the level of physiological HTT and/or of mutated and/oraggregated HTT and the reference standard indicates that the subject tobe diagnosed has a HTT amyloidosis or is at risk to develop a HTTamyloidosis. Preferably, the subject to be diagnosed and the controlsubject(s) are age-matched. The sample to be analyzed may be any bodyfluid suspected to contain pathologically mutated and/or aggregated HTT,for example a blood, blood plasma, blood serum, urine, peritoneal fluid,saliva or cerebral spinal fluid (CSF). In another aspect of the presentinvention, the antibodies of the present invention can be used indetection of soluble and aggregated HTT utilizing e.g. a TR-FRET basedduplex immunoassay as described in Baldo et al., Chem. Biol. 19(2)(2012), 264-275 which disclosure content, in particular the experimentalprocedures at pages 273-274, are incorporated herein.

Furthermore, it has been described in e.g. Ren et al., Nature Cell Biol.11 (2) (2009), 219-225 that mammalian cells can internalize fibrillarpolyglutamine peptide aggregates in culture gaining access to thecytosolic compartment and become co-sequestered in aggresomes togetherwith components of the ubiquitin-proteasome system and cytoplasmicchaperones. These internalized fibrillar aggregates were able toselectively recruit soluble cytoplasmic proteins and to confer aheritable phenotype upon cells expressing the homologous amyloidogenicprotein from a chromosomal locus. Therefore, in one embodiment of thepresent invention the anti-HTT antibody can reduce extracellularspreading or transneuronal propagation of “toxic” HTT species, as shownby Pecho-Vriesling et al. Nat. Neurosci. (2014) doi:10.1038/nn.3761 forhuntingtin or other proteins involved in neurodegeneration such asα-synuclein; see e.g. Guo et al., Nat Med. 20(2) (2014), 130-138.

The level of physiological HTT and/or of pathologically mutated and/oraggregated HTT may be assessed by any suitable method known in the artcomprising, e.g., analyzing HTT by one or more techniques chosen fromWestern blot, immunoprecipitation, enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting(FACS), two-dimensional gel electrophoresis, mass spectroscopy (MS),matrix-assisted laser desorption/ionization-time of flight-MS(MALDI-TOF), surface-enhanced laser desorption ionization-time of flight(SELDI-TOF), high performance liquid chromatography (HPLC), fast proteinliquid chromatography (FPLC), multidimensional liquid chromatography(LC) followed by tandem mass spectrometry (MS/MS), and laserdensitometry. Preferably, said in vivo imaging of HTT comprisesscintigraphy, positron emission tomography (PET), single photon emissiontomography (SPECT), near infrared (NIR) optical imaging or magneticresonance imaging (MRI).

In one embodiment thus, an antibody of the present invention or aHTT-binding molecule having substantially the same binding specificitiesof any one thereof, the polynucleotide, the vector or the cell asdefined hereinabove or a pharmaceutical or diagnostic compositioncomprising any one thereof is provided for use in prophylactictreatment, therapeutic treatment, and/or monitoring the progression or aresponse to treatment of a disease or disorder related to HTT. Ingeneral thus, the present invention also relates to a method ofdiagnosing or monitoring the progression of a disease or disorderrelated to HTT (such as HTT amyloidosis) in a subject, the methodcomprising determining the presence of HTT in a sample from the subjectto be diagnosed with at least one antibody of the present invention or aHTT-binding molecule having substantially the same binding specificitiesof any one thereof, wherein the presence of mutated, misfolded and/oraggregated HTT species or fragments thereof is indicative for thedisease or disorder. In one embodiment said method of diagnosing ormonitoring the progression of HTT amyloidosis in a subject is provided,the method comprising determining the presence of mutated and/oraggregated HTT and/or fragments thereof in a sample from the subject tobe diagnosed with at least one antibody of the present invention or aHTT-binding molecule having substantially the same binding specificitiesof any one thereof, wherein the presence of mutated and/or aggregatedHTT and/or fragment thereof is indicative of presymptomatic, prodromalor clinical HTT amyloidosis an increase of the level of HTT aggregatesin comparison to the level of the physiological HTT or in comparison toa reference sample derived from a healthy control subject or a controlsample from the same subject is indicative for progression ofpresymptomatic, prodromal or established HTT amyloidosis. It would beappreciated by any person skilled in the art that in one embodiment saidmethod is used as well for the diagnosing or monitoring the progressionof any other disease or disorder from the group of disorders related toHTT as defined hereinabove.

As indicated above, the antibodies of the present invention, fragmentsthereof and molecules of the same binding specificity as the antibodiesand fragments thereof may be used not only in vitro but in vivo as well,wherein besides diagnostic, therapeutic applications as well may bepursued. In one embodiment thus, the present invention also relates to aHTT binding molecule comprising at least one CDR of an antibody of thepresent invention for the preparation of a composition for in vivodetection of or targeting a therapeutic and/or diagnostic agent to HTTin the human or animal body. Potential therapeutic and/or diagnosticagents may be chosen from the nonexhaustive enumerations of thetherapeutic agents useful in treatment HTT amyloidosis and potentiallabels as indicated hereinbefore. In respect of the in vivo imaging, inone preferred embodiment the present invention provides said HTT bindingmolecule comprising at least one CDR of an antibody of the presentinvention, wherein said in vivo imaging comprises scintigraphy, positronemission tomography (PET), single photon emission tomography (SPECT),near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).In a further embodiment the present invention also provides saidHTT-binding molecule comprising at least one CDR of an antibody of thepresent invention, or said molecule for the preparation of a compositionfor the above specified in vivo imaging methods, for the use in themethod of diagnosing or monitoring the progression of a disease ordisorder related to HTT in a subject, as defined hereinabove.

VII. Peptides with Aggregation Specific HTT Epitopes

In a further aspect the present invention relates to peptides having anepitope of a polyP-rich region of HTT specifically recognized by anyantibody of the present invention. Preferably, such peptide comprises orconsists of an amino acid sequence as indicated in SEQ ID Nos.: 146,147, 148, 149, 150, 152, 153, 155, 156, 139, 151, 154, 158, 161, 157,159, 160 as the unique linear epitope recognized by the antibody or amodified sequence thereof in which one or more amino acids aresubstituted, deleted and/or added, wherein the peptide is recognized byany antibody of the present invention, preferably by antibodyNI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9,NI-302.37C12, NI-302.55D8, NI-302.78H12, NI-302.71F6, NI-302.33C11,NI-302.44D7, NI-302.7A8, NI-302.3D8, NI-302.46C9, NI-302.11H6,NI-302.18A1, NI-302.52C9, and/or NI-302.8F1.

In an additional aspect the present invention relates to peptides havingan epitope of the P-rich-region of HTT specifically recognized by anyantibody of the present invention. Preferably, such peptide comprises orconsists of an amino acid sequence as indicated in SEQ ID Nos. 140, 141,142, 143, 200 as the unique linear epitope recognized by the antibody ora modified sequence thereof in which one or more amino acids aresubstituted, deleted and/or added, wherein the peptide is recognized byany antibody of the present invention, preferably by antibodyNI-302.63F3, NI-302.31F11, NI-302.2A2, NI302.15D3 and/or NI-302.64E5.

Furthermore, in one embodiment the present invention relates to peptideshaving an epitope of the C-terminal region of HTT specificallyrecognized by any antibody of the present invention. Preferably, suchpeptide comprises or consists of an amino acid sequence as indicated inSEQ ID NO: 145 or SEQ ID NO: 202 as the unique linear epitope recognizedby the antibody or a modified sequence thereof in which one or moreamino acids are substituted, deleted and/or added, wherein the peptideis recognized by any antibody of the present invention, preferably byantibody NI-302.35C1 or NI-302.72F10.

In an additional aspect the present invention relates to peptides havingan epitope of the N-terminal-region of HTT specifically recognized byany antibody of the present invention. Preferably, such peptidecomprises or consists of an amino acid sequence as indicated in SEQ IDNOs: 144 as the unique linear epitope recognized by the antibody or amodified sequence thereof in which one or more amino acids aresubstituted, deleted and/or added, wherein the peptide is recognized byany antibody of the present invention, preferably by antibodyNI-302.15E8.

Furthermore, in one embodiment the present invention relates to peptideshaving an epitope of the Q/P-rich-region of HTT specifically recognizedby any antibody of the present invention. Preferably, such peptidecomprises or consists of an amino acid sequence as indicated in SEQ IDNO: 201 as the unique linear epitope recognized by the antibody or amodified sequence thereof in which one or more amino acids aresubstituted, deleted and/or added, wherein the peptide is recognized byany antibody of the present invention, preferably by antibodyNI-302.7D8.

In addition, in one embodiment the present invention relates to peptideshaving an epitope of HTT specifically recognized by any antibody of thepresent invention, preferably by antibody NI-302.6N9, NI-302.12H2,NI-302.8M1 and/or NI-302.4A6 in which one or more amino acids aresubstituted, deleted and/or added, wherein the peptide is recognized byany antibody of the present invention.

In one embodiment of this invention such a peptide may be used fordiagnosing or monitoring a disease or disorder related to mutated,misfolded and/or aggregated HTT species and/or fragments thereof in asubject, such as HD and/or HTT amyloidosis comprising a step ofdetermining the presence of an antibody that binds to a peptide in abiological sample of said subject, and being used for diagnosis of sucha disease in said subject by measuring the levels of antibodies whichrecognize the above described peptide of the present invention andcomparing the measurements to the levels which are found in healthysubjects of comparable age and gender. Thus in one embodiment thepresent invention relates to a method for diagnosing HTT amyloidosisindicative of presymptomatic or clinical HD in a subject, comprising astep of determining the presence of an antibody that binds to a peptideas defined above in a biological sample of said subject. According tothis method, an elevated level of measured antibodies specific for saidpeptide of the present invention is indicative for diagnosing in saidsubject presymptomatic or clinical HD or for diagnosing in said subjectany other disease or disorder from the group of disorders related to HTTas defined hereinabove. The peptide of the present invention may beformulated in an array, a kit and composition, respectively, asdescribed hereinbefore. In this context, the present invention alsorelates to a kit useful in the diagnosis or monitoring the progressionof HD and/or HTT amyloidosis, said kit comprising at least one antibodyof the present invention or a HTT-binding molecule having substantiallythe same binding specificities of any one thereof, the polynucleotide,the vector or the cell and/or the peptide as respectively definedhereinbefore, optionally with reagents and/or instructions for use.

The above disclosure generally describes the present invention. Unlessotherwise stated, a term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations may be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this applicationincluding the background section and manufacturer's specifications,instructions, etc.) are hereby expressly incorporated by reference;however, there is no admission that any document cited is indeed priorart as to the present invention.

A more complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES Example 1: Isolation and Identification of Anti-HTT Antibodies

Human-derived antibodies targeting HTT and/or mutated and/or aggregatedHTT species and/or fragments thereof were identified utilizing themethod described in the international application WO 2008/081008 thedisclosure content of which is incorporated herein by reference, withmodifications. In particular, wild-type and mutant HTT proteins obtainedby recombinant expression were used in both native andmutated-aggregated conformations for the identification of HTT-targetingantibodies. The mutated-aggregated conformations were produced in vitro,using a procedure similar to the one described in Example 3.

Example 2: Determination of Antibody Sequence

The amino acid sequences of the variable regions of the above identifiedanti-HTT antibodies were determined on the basis of their mRNAsequences, see FIG. 1. In brief, living B cells of selectednon-immortalized memory B cell cultures were harvested. Subsequently,the mRNAs from cells producing selected anti-HTT antibodies wereextracted and converted in cDNA, and the sequences encoding theantibody's variable regions were amplified by PCR, cloned into plasmidvectors and sequenced.

In brief, a combination of primers representing all sequence families ofthe human immunoglobulin germline repertoire was used for theamplifications of leader peptides, V-segments and J-segments. The firstround of amplification was performed using leader peptide-specificprimers in 5′-end and constant region-specific primers in 3′-end (Smithet al., Nat Protoc. 4 (2009), 372-384). For heavy chains and kappa lightchains, the second round of amplification was performed usingV-segment-specific primers at the 5′-end and J-segment-specific primersat the 3′-end. For lambda light chains, the second round amplificationwas performed using V-segment-specific primers at the 5′-end and aC-region-specific primer at the 3′-end (Marks et al., Mol. Biol. 222(1991), 581-597; de Haard et al., J. Biol. Chem. 26 (1999),18218-18230).

Identification of the antibody clone with the desired specificity wasperformed by re-screening on ELISA upon recombinant expression ofcomplete antibodies. Recombinant expression of complete human IgG1antibodies was achieved upon insertion of the variable heavy and lightchain sequences “in the correct reading frame” into expression vectorsthat complement the variable region sequence with a sequence encoding aleader peptide at the 5′-end and at the 3′-end with a sequence encodingthe appropriate constant domain(s). To that end the primers containedrestriction sites designed to facilitate cloning of the variable heavyand light chain sequences into antibody expression vectors. Heavy chainimmunoglobulins were expressed by inserting the immunoglobulin heavychain RT-PCR product in frame into a heavy chain expression vectorbearing a signal peptide and the constant domains of human or mouseimmunoglobulin gamma 1. Kappa light chain immunoglobulins were expressedby inserting the kappa light chain RT-PCR-product in frame into a lightchain expression vector providing a signal peptide and the constantdomain of human kappa light chain immunoglobulin. Lambda light chainimmunoglobulins were expressed by inserting the lambda light chainRT-PCR-product in frame into a lambda light chain expression vectorproviding a signal peptide and the constant domain of human or mouselambda light chain immunoglobulin.

Functional recombinant monoclonal antibodies were obtained uponco-transfection into HEK 293 or CHO cells (or any other appropriaterecipient cell line of human or mouse origin) of an Ig-heavy-chainexpression vector and a kappa or lambda Ig-light-chain expressionvector. Recombinant human monoclonal antibody was subsequently purifiedfrom the conditioned medium using a standard Protein A columnpurification. Recombinant human monoclonal antibody can produced inunlimited quantities using either transiently or stably transfectedcells. Cell lines producing recombinant human monoclonal antibody can beestablished either by using the Ig-expression vectors directly or byre-cloning of Ig-variable regions into different expression vectors.Derivatives such as F(ab), F(ab)₂ and scFv can also be generated fromthese Ig-variable regions. The framework and complementarity determiningregions were determined by comparison with reference antibody sequencesavailable in databases such as Abysis (www.bioinf.org.uk/abysis/) and(www.imgt.org/), and annotated using the Kabat numbering scheme(www.bioinf.org.uk/abs/).

Example 3: Expression of HTT Exon 1 Proteins Methods

Recombinant huntingtin exon1 proteins GST-HttExon1Q21 (GST-HD21),GST-HttExon1Q35 (GSTHD35), and GST-HttExon1Q49 (GST-HD49) expression andpurification pGEX-6P-1 expression vector (GE Healthcare) encoding Exon1of human huntingtin with polyQ length of 21, 35 or 49 CAG repeats,respectively (compare FIG. 2A) fused with a PreScission cleavage site toan N-terminal Glutathione S-transferases (GST)-tag were expressed in E.coli strain BL21. Overnight bacterial cultures (37° C., 220 rpm) werediluted 1:25 and expression was induced at an Absorption 600 of 0.5-0.6for 4 hrs by addition of 1 mM IPTG (Sigma 11284) and further incubationat 36° C., 220 rpm. Cultures were grown in LB medium containing 100μg/ml ampicillin at 37° C., for overnight cultures in addition with 1%glucose. Recombinant GST-HttExon1 proteins were purified by binding toglutathione agarose (Sigma G4510). Briefly, the bacteria pellet wasresuspended in 20-40 ml of cold buffer 1 (50 mM NaH2PO4, 5 mM Tris, 150mM NaCl, 1 mM EDTA pH8, 5 mg/ml final lysozyme, protease inhibitorcomplete (Roche)) were incubated for 60 min on ice, ultrasonicated,Triton-X100 added (0.1% final) and centrifuged for 90 min at 14,000 gafter incubation on ice for 5 min. Glutathione agarose was added to thesupernatant, incubated for 2 hrs at 4° C., spun down for 10 min at 1000g and washed 2× with cold PBS after removal of the supernatant. Elutionwas performed for 5 min in 1 ml buffer 1 with 10 mM reduced glutathionepH 9. This step was repeated 5 to 15 times until no further protein waseluted. The pooled supernatants were dialyzed against buffer (50 mM trispH7.4, 150 mM NaCl, 1 mM EDTA, 1% glycerol) over night (10 kD MWCO,Pierce) and aliquots were stored at −80° C.

SDS-PAGE Analysis

Purified recombinant GST-HttEx1 proteins were resolved by gradientSDS-PAGE (NuPAGE Bis-Tris 4-12%; Invitrogen, Basel, Switzerland)followed by staining with Coomassie brilliant blue or electroblotting onnitrocellulose membranes. Blots were incubated with primary antibodiesMab 5492 (Chemicon N-terminal aa1-82 epitope, P-rich domain) orNI-302.37C12 followed by a goat antimouse IgG secondary antibodyconjugated with HRP or donkey anti-human IgG secondary antibodyconjugated with HRP. Blots were developed using ECL and ImageQuant LAS4000 detection (GE Healthcare, Switzerland).

As shown in FIG. 2B the different recombinant GST-HttEx1 proteins weresuccessfully expressed and purified as demonstrated by Coomassiestaining after SDS-PAGE.

Example 4: Characterization of Aggregation State by Dot Blot and FilterRetardation

To characterize HD21, HD35 and HD49 protein aggregation kinetics filterretardation and dot-blot analyses were performed.

Therefore, at the beginning an aggregation reaction was performed asfollows: Recombinant GST-HttExon1 proteins can be expressed and purifiedas a fusion protein. As soon as the GST tag is cleaved off from thefusion protein by the PreScission Protease (PP) the aggregation reactionof the huntingtin Exon1 protein starts immediately. Before the start ofreactions the GST-HttExon1 proteins were centrifuged at 100,000 g for 30minutes. The cleared protein solution were diluted to 2 μM proteinconcentration in cold aggregation buffer (0.05 M Tris/HCL pH 7, 0.15 MNaCl, 1 mM EDTA) and 1 mM DTT and PreScission Protease (GE Healthcare)were added. The reaction was incubated at room temperature with rotatingat 300 rpm and the aggregation reactions were stopped by snap freezingat −80° C. after the indicated time intervals. Aliquots of HD21, HD35and HD49 aggregation reactions were subsequently removed after 1, 3, 5,7 and 24 hrs of incubation time, respectively, snap frozen on dry iceand stored at −80° C.

For the dot blot analysis samples were thawed on ice, diluted andtransferred onto a nitrocellulose transfer membrane with a filter deviceapplying vacuum in the chamber below the membrane. To that end, themembrane was equilibrated with PBS, mounted in the chamber and washedwith 100 μl PBS per well. The samples were loaded and completely suckedthrough the membrane followed by 3 washes with PBS. The device wasdissembled and the membrane was briefly air-dried for 15 min at roomtemperature, blocked for 1 hour at room temperature with blocking buffer(3% BSA, 0.1% Tween 20 in PBS buffer) and incubated with polyclonal HD-1antibody (1:10,000, kind gift of Prof. E. Wanker, MDC, Berlin). Afterwashing, the membrane was incubated for 1 h at RT with an anti-rabbitIgG antibody coupled to HRP and blots were developed using ECL andImageQuant LAS 4000 detection (GE Healthcare, Switzerland).

As evident from the dot blot shown in FIG. 2C, left side, polyclonalHD-1 antibody detected HD21, HD35 and HD49 proteins irrespective oftheir aggregation state.

For filter retardation assays samples were thawed on ice, diluted indenaturation buffer (4% SDS, 100 mM DTT) and transferred through acellulose acetate membrane with a pore size of 0.2 um using a vacuumchamber: To that end, the membrane was equilibrated in 0.1% SDS in PBS,mounted on the vacuum chamber and the wells were washed with 0.1% SDS.The samples were added, filtered through the membrane by vacuum andwashed 3 times with 0.1% SDS. The membrane was then removed from thechamber, briefly air-dried, blocked for 1h at RT in blocking buffer (5%milk, 0.1% Tween 20 in PBS buffer), incubated with polyclonal HD-1antibody (1:5,000, Scherzinger et al., Cell 90 (1997), 549-558) andprocessed further as described above.

In the filter retardation assay, the first aggregates retained by themembrane were detected for HD35 after 24 hours of incubation. HD49proteins with an extended polyQ tract form insoluble aggregated as earlyas 3 hrs after cleavage of the GST tag, see FIG. 2C right side (FRA).

Example 5: Characterization of Huntingtin Exon1 Aggregates

To verify and characterize HD35 and HD49 Exon1 aggregate formationelectron microscopy (EM) was performed. In brief, HD49 aggregationreactions after 1, 3 and 24 hrs, respectively or samples from HD35 after24 hrs were analyzed by electron microscopy. Samples were adsorbed ontoglow-discharged carboncoated copper grids. Excess sample was removed byblotting on filter paper. Grids were stained with 2% (w/v) uranylacetate for 1 min and excess uranyl acetate was washed with distilleddeionized water. Grids were air-dried and imaged using a Philips CM100transmission electron microscope with an acceleration voltage of 100 kV.

EM analysis of the HD35 aggregation reaction revealed larger aggregatesvisible by EM resembling protofibrillar structures after 24 hrs ofincubation (FIG. 2D [E]). HD49 displayed a more rapid aggregationkinetics with fibrils being detectable already after 1 hour ofincubation (FIG. 2D [F]) and increasing in size and number withaggregation time (FIG. 2D [C, D, G, H]). These observations wereconsistent with the results obtained in the filter retardation assayswhere aggregates larger than 0.2 m are retained on the cellulose acetatemembrane and confirm the successful preparation of huntintingtin exon 1aggregates; see also Example 4.

Example 6: Binding Affinity of Anti-polyP Domain NI-302.33C11 AntibodyUtilizing Direct ELISA and EC₅₀

To determine the half maximal effective concentration (EC₅₀) ofrecombinant human-derived HTT antibody NT-302.33C11 to soluble andaggregated huntingtin Exon1 proteins with 21 or 49 polyQ repeats directELISA was performed. In brief, 96 well microplates (Corning) were coatedwith either GST-HD21, GST-HD49 or aggregated HD21 or HD49 at aconcentration of 5 μg/ml in coating buffer (15 mM Na2CO3, 35 mM NaHCO₃,pH 9.42). Nonspecific binding sites were blocked for 1 h at RT withPBS/0.1% Tween®-20 containing 2% BSA (Sigma-Aldrich, Buchs,Switzerland). Primary antibodies were diluted to the indicatedconcentrations and incubated 1 h at RT. Binding was determined usingeither a donkey anti-human IgG Fcγ-specific antibody conjugated with HRPor a goat anti-mouse IgG (H+L)-specific antibody conjugated with HRP,followed by measurement of HRP activity in a standard colorimetricassay. Subsequently, EC₅₀ values were estimated by a non-linearregression using GraphPad Prism software (San Diego, USA).

The EC₅₀ of human-derived HTT antibody NI-302.33C11 for aggregated andsoluble HTT exon 1 proteins with 21 or 49 poly Q repeats was determinedby direct ELISA with coating of the different preparations at 5 μg/mlconcentration. As shown in FIGS. 3A and B antibody NI-302.33C11 boundwith similar high affinity to all four species including thepathologically aggregated HTT Exon1 HD49 with an EC50 of approximately100 pM.

Example 7: Binding Selectivity of Anti-HTT Antibodies Utilizing Dot Blotand Filter Retardation Assay

To characterize recombinant human-derived HTT antibody NI-302.33C11 tosoluble and aggregated huntingtin Exon1 proteins with 21, 35 or 49 polyQrepeats filter retardation assay and dot-blot were performed. For thisreason, aliquots of HD21, HD35 and HD49 aggregation reactions asdescribed in Example 4 were removed after 1, 3, 5, 7 and 24 hrs ofincubation time, snap frozen on dry ice and stored at −80° C. and a dotblot was performed as described in Example 4. Filter retardation assaywas also performed as described in Example 4, with the exception thatthe membrane was incubated with NI-302.33C11 (1 μg/ml).

It could be shown that on the dot blot (FIG. 3C, left side), antibodyNI-302.33C11 preferentially detects proteins of huntingtin with expandedpolyQ tracts (HD49>>HD35>HD21). Furthermore, the signal intensityincreased with increasing incubation times of the aggregation reactionsof HD35 and HD49.

This is also true for the results shown in the filter retardationanalysis (FIG. 3C, right side), which showed that NI-302.33C11 detectsHD35 and HD49 aggregates that were retained on the 0.2 μm pore sizemembrane. These findings based on spotted protein preparations suggestedthat antibody NI-302.33C11 has a preference for aggregated HTTconformations with pathogenic polyQ expansions.

Example 8: Binding Specificity and Selectivity of Anti-HTT Antibodies toUnrelated Aggregating Protein Targets Utilizing Direct ELISA

To determine the binding specificity antibody NI-302.33C11 recombinantantibody binding to the polyP-region of HTT and not to unrelatedaggregating protein targets direct ELISA was performed on 96 wellmicroplates (Corning) coated with different target proteins at aconcentration of 1-10 μg/ml in coating buffer (15 mM Na2CO3, 35 mMNaHCO₃, pH 9.42). Non-specific binding sites were blocked for 1 h at RTwith PBS/0.1% Tween®-20 containing 2% BSA (Sigma-Aldrich, Buchs,Switzerland). NI-302.33C11 antibody was diluted to the indicatedconcentrations and incubated 1 h at RT. Binding was determined usingdonkey anti-human IgG Fcγ-specific antibody conjugated with HRP followedby measurement of HRP activity in a standard colorimetric assay. Signalsfor target protein were calculated in fold increase above median.

It could be shown that human-derived NI-302.33C11 binds specifically toHTT, i.e. aggregated HD49, with absent binding to the other unrelatedprotein targets including prominent amyloid-forming proteins, see FIG.16A.

Example 9: Assessment of the Binding Epitope of the HTT AntibodyNI-302.33C11

To map the huntingtin (HTT) epitope recognized by the NI-302.33C11human-derived antibody epitope mapping by peptide scanning analysis withsynthetic peptides was performed.

In brief, scans of overlapping peptides were used for epitope mapping.The sequence of human HTT Exon 1 sequence was synthesized as a total of16 linear 15-meric peptides with 10 aa overlap between individualpeptides (JPT Peptide Technologies, Berlin, Germany) and spotted ontonitrocellulose membranes. The membrane was activated for 5 min inmethanol and then washed at RT in TBS for 10 min. Non-specific bindingsites were blocked for 2 hours at room temperature with Roti®-Block(Carl Roth GmbH+Co. KG, Karlsruhe, Germany). Human NI-302.33C11 antibody(1 μg/ml) was incubated for 3 hrs at RT in Roti®-Block. Binding ofprimary antibody was determined using HRP conjugated donkey-anti humanIgGγ secondary antibody. Blots were developed using ECL and ImageQuantLAS 4000 detection (GE Healthcare, Switzerland).

As shown in FIG. 4, prominent binding of NI-302.33C11 was observed topeptides number 7, 8, 9, 13 and 14 indicating that the epitoperecognized by this antibody is localized in the polyP repeat domain ofhuntingtin. The NI-302.33C11 binding epitope is therefore predicted tobe localized within HTT amino acids 35-PPPPPPPP-42 (SEQ ID No.: 139) andamino acids 63-PPPPPPPPPPP-72 (SEQ ID No.: 162).

Example 10: Epitope Mapping by Direct ELISA Binding to Different Exon1Peptides of the HTT Antibody NI-302.33C11

To determine the half maximal effective concentration (EC₅₀) ofrecombinant human-derived HTT antibody NI-302.33C11 to BSA-coupledpeptide fragments of the huntingtin Exon 1 direct ELISA with BSA-coupledHtt Exon1 domain peptides was performed.

In brief, 96 well microplates (Corning) were coated with BSA-coupledsynthetic peptides (Schafer-N, Denmark) of the N-terminal amino acid1-19 (MATLEKLMKAFESLKSFQQ, SEQ ID No.: 93), the P-rich domain sequence(PPQLPQPPPQAQPLLPQPQPP, SEQ ID No.: 94), the polyP repeat sequence(PPPPPPPPPPP, SEQ ID No.: 95) or the 14 C-terminal amino acids(PPGPAVAEEPLHRP, SEQ ID No.: 96) or with the full lengths GST-HD49 Exon1 protein at 5 g/ml in coating buffer (15 mM Na2CO3, 35 mM NaHCO₃, pH9.42). Non-specific binding sites were blocked for 1 h at RT withPBS/0.1% Tween®-20 containing 2% BSA (Sigma-Aldrich, Buchs,Switzerland). Primary antibodies were diluted to the indicatedconcentrations and incubated 1 h at RT. Binding was determined usingdonkey anti-human IgG Fcγ-specific antibody conjugated with HRP,followed by measurement of HRP activity in a standard colorimetric assayand the EC₅₀ values were estimated by a non-linear regression usingGraphPad Prism software (San Diego, USA).

As shown in FIG. 5 NI-302.33C11 bound with high affinity to theBSA-coupled polyP peptide as well as to full-length GST-HD49 with anequivalent EC₅₀ of 30 pM. This confirms the epitope mapping to the polyPsequence as shown in Example 9.

Example 11: Assessment of the Purity and Integrity of Recombinant HumanNI-302.33C11 Anti-polyP Domain Antibody

To assess the purity and integrity of recombinant human NI-302.33C11anti-polyP domain lead antibody human NI-302.33C11 anti-polyP domainantibody was expressed by transient transfection of CHO-S cells andpurified by protein A affinity purification on an Akta system.

After PD-10 column desalting the antibody was formulated in PBS.Subsequently SDS-PAGE analysis was performed, wherein two and 10 μg ofpurified recombinant human NI-302.33C11 anti-polyP domain antibody wereresolved under reducing conditions by gradient SDS-PAGE (NuPAGE 4-12%Bis-Tris gel; Invitrogen) followed by Coomassie staining (SimplyBlueSafeStain, Invitrogen).

The SDS-PAGE analysis under reducing conditions of the recombinant humanNI-302 anti-polyP domain lead antibody revealed two major bandscorresponding to the antibody heavy and light chains at the expectedsize as shown in FIG. 6, while no significant contaminations orproteolytic degradation products were detected.

Example 12: Characterization of HTT Antibody NI-302.33C11 in Human HTTTransgenic Mice

To assess the binding of NI-302.33C11 antibody to huntingtin pathologyin human HTT transgenic mouse brain tissues immunohistochemistry wasperformed. The B6.Cg-Tg(HDexon1)61Gpb/J transgenic mouse line(Mangiarini et al., Cell 87 (1996), 493-506) is a well characterizedmouse model for Huntington's Disease (HD). Starting at around 9 weeks ofage, this animal model develops a progressive pathology characterized byintranuclear inclusions of huntingtin reminiscent of human Huntington'sdisease (Naver et al., Neuroscience 122 (2003), 1049-1057). Hemibrainsof these B6.Cg-Tg(HDexon1)61Gpb/J transgenic mice at a progressed stageof disease (270 days) were fixed in phosphate-buffered 4%paraformaldehyde solution, paraffin-embedded, and 5-μm sections wereprepared. After formic acid and citrate buffer pretreatment, sectionswere incubated with 1, 5 or 50 nM human NI-302.33C11 anti-HTT antibodyfollowed by incubation with biotinylated donkey-anti-human secondaryantibody (Jackson Immunoresearch; 1:250). Antibody signal was amplifiedwith the Vectastain ABC kit (Vector Laboratories) and detected withdiaminobenzidine (Pierce). As shown in FIG. 27 human-derived polyPdomain antibody NI-302.33C11 revealed a very prominent staining ofneuronal intranuclear inclusion pathology already at the lowest 1 nMconcentration (FIG. 27 [E-H]) consistent with the high affinity bindingto huntingtin aggregates as determined by ELISA and dot blot analyses.At a concentration of 5 nM or higher, the antibody stained in additionentire medium spiny neurons and produced a more generalized diffusestaining which was also detectable on nontransgenic brain sections. Acertain degree of cross-reactivity cannot be excluded as the polypepitope targeted by NI-302.33C11 was present also in numerous unrelatedproteins (FIG. 27 [F-H]).

Example 13: Characterization of Binding Affinity and Selectivity ofAnti-P-Rich Domain NI-302.63F3 Antibody Utilizing Direct ELISA and EC₅₀

To determine the half maximal effective concentration (EC₅₀) ofrecombinant human-derived HTT antibody NI-302.63F3 to soluble andaggregated HTT Exon1 proteins with 21 or 49 polyQ repeats direct ELISAand EC50 determination was performed as described in Example 6, supra.

It could be shown that NI-302.63F3 binds with similar high affinity toall four species including the aggregated HTT Exon1 HD49 with an EC50 ofapproximately 200 to 400 pM FIGS. 7 A and B. Accordingly, thehuman-derived HTT anti-P-rich domain antibody NI-302.63F3 targets anepitope exposed in aggregated as well as in an uncut, more linearstructure of HTT Exon1 protein with high-affinity in the subnanomolarrange.

Additionally, to characterize the binding of recombinant human-derivedHTT antibody NI-302.63F3 to soluble and aggregated HTT Exon1 proteinswith 21, 35 or 49 polyQ repeats using filter retardation assay and dotblot as described in Example 7, supra, with the small modification thatthe incubation was performed with 1 μg/ml of NI-302.63F3 antibody.

On the dot blot, antibody NI-302.63F3 most prominently detected the HD49protein with an expanded polyQ tract (FIG. 7C, left side). In the filterretardation analysis NI-302.63F3 detected HD35 and HD49 aggregates thatwere retained on the 0.2 μm pore size membrane (FIG. 7C, right side).These findings based on spotted protein preparations demonstrate thatantibody NI-302.63F3 recognizes aggregated HTT conformations withpathogenic polyQ expansions.

Furthermore, to determine the binding of NI-302.63F3 recombinantantibody to unrelated aggregating protein targets, direct ELISA wasperformed as described in Example 8, supra. As shown in FIG. 16Bhuman-derived NI-302.63F3 bound specifically to HTT while a binding tounrelated proteins could not be shown.

Example 14: Assessment of the Binding Epitope of the HTT AntibodyNI-302.63F3

To map the huntingtin epitope recognized by the NI-302.63F3human-derived antibody epitope mapping with synthetic peptides wasperformed as described above in Example 9.

As shown in FIG. 8, prominent binding of NI-302.63F3 was observed topeptides number 10 and 11 with a weak signal on peptide 8 and 9indicating that the epitope recognized by this antibody is localized inthe P-rich domain (between the polyP repeat regions) of HTT. TheNI-302.63F3 binding epitope was therefore predicted to be localizedwithin HTT amino acid sequence 43-(PPPQL)PQPPPQAQPL-57 (SEQ ID Nos.: 161and 140).

Example 15: Epitope Mapping by Direct ELISA Binding to Different Exon1Peptides of the HTT Antibody NI-302.63F3

To determine the half maximal effective concentration (EC50) ofrecombinant human-derived HTT antibody NI-302.63F3 to BSA-coupledpeptide fragments of the huntingtin Exon 1 direct ELISA with BSA-coupledHtt Exon1 domain peptides and EC₅₀ determination were performed asdescribed in Example 10.

As shown in FIG. 9 NI-302.63F3 binds with high affinity to theBSA-coupled P-rich domain peptide as well as to full-length GST-HD49with a similar EC₅₀ of 200 to 300 pM. This confirms the epitope mappingto the P-rich domain sequence as shown in Example 14.

Example 16: Assessment of the Purity and Integrity of Recombinant HumanNI-302.63F3 Anti-P-Rich Domain Antibody

To assess the purity and integrity of recombinant human NI-302.63F3anti-proline-rich domain antibody SDS-PAGE analysis was performed asalready described in Example 11, supra.

SDS-PAGE analysis under reducing conditions of the recombinant humanNI-302.63F3 anti-P-rich domain antibody revealed two major bandscorresponding to the antibody heavy and light chains at the expectedsize. No significant contaminations or proteolytic degradation productswere detected as shown in FIG. 10.

Example 17: Characterization of HTT Antibody NI-302.63F3 in Human HTTTransgenic Mice

The assessment of the binding of NI-302.63F3 anti-P-rich domain antibodyto HTT pathology in human HTT transgenic mouse brain tissues wasassessed as described in Example 12, supra with the difference that theincubation of the sections was performed with 1 or 50 nM of theanti-P-rich domain antibody.

As shown in FIG. 28 [E-F] the human-derived NI-302.63F3 anti-P-richdomain antibody revealed a prominent and highly specific staining ofneuronal intranuclear inclusion pathology at 1 and 50 nM concentrationin the striatum and cortex of R6/1 transgenic animals consistent withthe high affinity binding to HTT aggregates as determined by ELISA anddot blot analysis. However as shown in FIG. 28 [F] at a concentration of50 nM the antibody NI-302.63F3 stained additionally weakly the entirenucleus of the medium spiny neurons.

Example 18: Characterization of Binding Affinity and Selectivity ofAnti-C-Terminal Domain Antibodies NI-302.35C1 and NI-302.72F10 UtilizingDirect ELISA and EC₅₀

To determine the half maximal effective concentration (EC₅₀) ofrecombinant human-derived HTT antibodies NI-302.35C1 and NI-302.72F10 tosoluble and aggregated HTT Exon1 proteins with 21 or 49 polyQ repeatsdirect ELISA and EC50 determination was performed as described inExample 6, supra.

It could be shown that NI-302.35C1 binds with high affinity to all fourspecies including the aggregated HTT Exon1 HD49 with an EC₅₀ ofapproximately 2.7 nM; see FIG. 11 A and B. Similarly, NI-302.72F10 bindsto all four species albeit with a different affinity than NI-302.35C1(aggregated HD21>>GST-HD21>>aggregated HD49>>GST-HD49) (FIG. 31 C) whichmay be explained with the different epitopes recognized by bothantibodies (FIG. 20).

Accordingly, the human-derived HTT anti-C-terminal domain antibodiesNI-302.35C1 and NI-302.72F10 target an epitope exposed in aggregated aswell as in soluble forms of HTT with low nanomolar affinity.

Additionally, to characterize the binding of recombinant human-derivedHTT antibodies NI-302.35C1 and NI-302.72F10 to soluble and aggregatedHTT Exon1 proteins with 21, 35 or 49 polyQ repeats filter retardationassay and dot blot as described in Example 7, supra, was performed.

On the dot blot, antibody NI-302.35C1 preferentially detected constructsof HTT with expanded polyQ tracts (HD49>HD35>>HD21, FIG. 11C, leftside). Furthermore, the signal intensity increases with increasingincubation times of the aggregation reactions of HD35 and HD49.Similarly, antibody NT-302.72F10 detected constructs of HTT withexpanded polyQ tracts albeit with a different preference(HD35>>HD49>HD21) whereas the signal intensity increases with increasingincubation times of the aggregation reactions of HD35 only (FIG. 32 C).

In the filter retardation analysis NI-302.35C1 detected HD35 and HD49aggregates that were retained on the 0.2 μm pore size membrane (FIG. 11C, right side) whereas NI-302.72F10 detected HD35 aggregates only (FIG.32 C, right side). These findings based on membrane bound proteinpreparations suggested that antibodies NI-302.35C1 and NI-302.72F10preferentially targets aggregated HTT conformations with pathogenicpolyQ expansions.

Furthermore, to determine the binding of NI-302.35C1 and NI-302.72F10recombinant antibodies to unrelated aggregating protein targets, directELISA was performed as described in Example 8, supra. As shown in FIG.16 C human-derived NI-302.35C1 as well as shown in FIG. 33 Chuman-derived NI-302.72F10 bound specifically to HTT while a binding tounrelated proteins could not be shown.

Example 19: Assessment of the Binding Epitope of the HTT AntibodyNI-302.35C1

To map the huntingtin epitope recognized by the NI-302.35C1human-derived antibody epitope mapping with synthetic peptides wasperformed as described above in Example 9.

Determination of NI-302.35C1 antibody binding epitope by scan ofoverlapping peptides did not result in specific signal. Therefore,processing this antibody in the way it was done for the other HTT NI-302antibodies did not results in any specific signal on the individualpeptides for unknown reasons. The epitope was successfully mapped to aC-terminal peptide by coupling it to BSA, see also Example 20.

Example 20: Epitope Mapping by Direct ELISA Binding to Different Exon1Peptides of the C-Terminal Domain HTT Antibody NI-302.35C1

To determine the half maximal effective concentration (EC₅₀) ofrecombinant human-derived HTT antibody NI-302.35C1 to BSA-coupledpeptide fragments of the HTT Exon 1 direct ELISA with BSA-coupled HttExon1 domain peptides and EC₅₀ determination were performed as describedin Example 10.

As shown in FIG. 12 NI-302.35C1 binds with high affinity to theBSA-coupled C-terminal peptide as well as to full-length GST-HD49 withan EC₅₀ value of approximately 0.7 nM and 3.2 nM, respectively. Thislocates the epitope to the C-terminal region of HTT Exon1 sequence(71-PPGPAVAEEPLHRP-85, SEQ ID No: 96). If the same C-terminal peptidewas coated directly to the plate only weak binding was observed(EC₅₀>100 nM, data not shown) suggesting that the presentation of thepeptide coupled to BSA increases the binding to the epitope and might bean explanation why the epitope mapping by peptide scanning analysis asshown in Example 20 did not work.

Example 21: Assessment of the Purity and Integrity of Recombinant HumanNI-302.35C1 Anti-P-Rich Domain Antibody

To assess the purity and integrity of recombinant human NI-302.35C1anti-C-terminal domain antibody SDS-PAGE analysis was performed asalready described in Example 11, supra.

SDS-PAGE analysis under reducing conditions of the recombinant humanNI-302.35C1 anti-C-terminal domain antibody revealed two major bandscorresponding to the antibody heavy and light chains at the expectedsize, while no significant contaminations or proteolytic degradationproducts were detected (FIG. 13).

Example 22: Characterization of HTT Antibody NI-302.35C1 in Human HTTTransgenic Mice

The assessment of the binding of NI-302.35C1 anti-C-terminal domainantibody to HTT pathology in human HTT transgenic mouse brain tissueswas assessed as described in Example 12, supra with the difference thatthe incubation of the sections was performed with 5 or 50 nM of theanti-C-terminal domain antibody.

As shown in FIG. 29 [E-F] the human-derived NI-302.35C1 anti-C-terminaldomain antibody revealed a prominent staining of neuronal intranuclearinclusion pathology at 5 and 50 nM concentration in striatum of R6/1transgenic animals consistent with the high affinity binding to HTTaggregates as determined by ELISA and dot-blot analyses.

Example 23: Assessment of the Effects of Human-Derived AntibodiesTargeting HTT on Spine Density in Hippocampal Slice Cultures AntibodyExpression and Purification

Human-derived antibodies targeting distinct domains in HTT exon 1 wereexpressed by transient transfection of CHO-S cells and purified byprotein A affinity purification on the Akta system. After PD-10 columndesalting the antibodies were formulated in PBS. Endotoxin levels wereconfirmed to be <10 EU/ml.

Hippocampal Slice Culture

Organotypic hippocampal slice cultures were prepared and culturedaccording to Stoppini et al., J Neurosci Methods. (1991) 37(2):173-82.In short, 6- to 8-d-old B6CBA-Tg(HDexon1)62Gpb/1J transgenic andnontransgenic littermates were decapitated, brains were removed, andboth hippocampi were isolated and cut into 400-μm thick slices. Thismethod yields thin slices which remain 1-4 cell layers thick and arecharacterized by a well preserved organotypic organization. Slices werecultured on Millicell culture plate inserts (0.4 m, Millipore) insix-well plates containing 1 ml of culture medium (46% minimum essentialmedium Eagle with HEPES modification, 25% basal medium with Earle'smodification, 25% heat-inactivated horse serum, 2 mM glutamine, 0.6%glucose, pH 7.2). Culture plates were kept at 37° C. in a humidifiedatmosphere containing 5% CO2. Slices were kept in culture for 7 d beforethe experiments. Culture medium was exchanged every second or third day.On day 7 antibodies were added at a concentration of 10 g/ml. On day 10in vitro slice cultures were infected with Sindbis virus using a dropletmethod (Shahani et al., J Neurosci. 31 (2006), 6103-6114). For spineanalysis, cultures were fixed at day 4 post infection (14 days invitro). Slices were left attached to the culture plate membrane topreserve hippocampal structure and rinsed with PBS. Slices were thenfixed with 4% paraformaldehyde in PBS containing 4% sucrose for 2 h at4° C. For each dendrite a picture was taken and spines were analyzedover a length of 30-m. Eight to 13 slices per group from a total of 12transgenic animals were quantified for each antibody treatment. Datarepresent the mean±SEM. *p<0.05 (MWU), #p=0.05.

The quantification of dendritic spine density in hipppocampal slicecultures of Tg(HDexon1)62Gpb/1J transgenic mice (FIG. 17 B, D) revealeda significant reduction by 53% compared to non-transgenic littermates(FIG. 17 A, C, E) using hippocampal slices of postnatal day 6 animalsafter 14 days in vitro with the experimental design described above.Upon addition of human-derived NT-302 antibodies at concentration of 10μg/ml for seven days a significant attenuation of spine density loss wasobserved for antibodies NI-302.31F11 (FIG. 17 F, p<0.05, t-test) andNI-302.63F3 (p=0.05, t-test), whereas antibody NI-302.33C11 andNI-302.35C1 did not show a clear effect under the conditions tested.

The significant reduction of spine density compared to non-transgeniclittermate in displayed in hippocampal slice cultures ofTg(HDexon1)62Gpb/1J transgenic mice led to the suggestion that this is asuitable model for in vitro testing of HTT candidate antibodies fortheir activity towards interference with HTT toxicity. In this model,antibodies NI-302.63F3 and NI-302.11F11 that both targeted the P-richdomain within HTT exon 1 were able to improved spine density compared toan isotype control antibody. This suggests that these antibodies canattenuate the toxic effects on spine density driven by expression ofpathological poly-Q-expanded HTT.

Example 24: Penetration of NI-302 Antibodies in the Brain of R6/1 AnimalModel

To test the penetration of the anti-HTT antibodies of the presentinvention a transgenic mice model was utilized. In particular,Tg(HDexon1)61Gpb/J transgenic mice harbor a 1.9 kb transgene which wasisolated from a phage genomic clone derived from an Huntington's disease(HD) patient and contained the 5′ end of the human huntingtin (HTT)gene. It was composed of approximately 1 kb of 5′ UTR sequences, exon 1(carrying expanded CAG repeats of ˜130 units) and the first 262 bp ofintron 1. This construct was microinjected into single cell CBAxC57BL/6embryos. Male founder R6 was bred to CBAxC57BL/6 females, producingseveral founder lines. Mice from founder line R6/1 have the transgeneintegrated as a single intact copy which is ubiquitously expressed.Transgenic mice on a mixed CBAxC57BL/6 genetic background werebackcrossed to C57BL/6J for more than 12 to generate the congenic strainB6.Cg-Tg(HDexon1)61Gpb/J.

R6/1 transgenic mice exhibit a progressive neurological phenotype thatmimics many of the features of HD, including choreiform-like movements,involuntary stereotypic movements, tremor, and epileptic seizures, aswell as nonmovement disorder components, including unusual vocalization.They urinate frequently and exhibit loss of body weight and muscle bulkthrough the course of the disease. Neurologically they develop neuronalintranuclear inclusions (NII) which contain both the HTT and ubiquitinproteins. These NII have also been identified in human HD patients. Theage of onset of HD symptoms is reported to occur between 15 and 21 weeksfor this 6/1 line.

The study animals displayed the following properties and were identifiedby classical ear marking:

Strain: Hemizygous B6.Cg-Tg(HDexon1)61Gpb/J (Mangiarini et al., Cell, 87(1996), 493-506)

Source: Jackson Laboratory, Maine, USA

Sex: Males and females

Age start: 230 to 260 days

Cohorts: NI-302.31F11 Total: 3 males

NI-302.35C1 Total: 3 males

Vehicle Total: 3 males

For the spinal cord homogenization B6.Cg-Tg(HDexon1)61Gpb/J transgenicmice were deeply anesthetized and transcardially perfused with coldphosphate-buffered saline through the left ventricle by mean of aperistaltic pump. The brain was dissected out and snap frozen on dryice. The tissue samples were homogenized in 1:10 w/v DEA-Buffer (50 mMNaCl, 0.2% DEA, protease inhibitor complete, Roche Diagnostics) with ahand-sonicator (Sartorius, Labsonic M). Samples were centrifuged for 30minutes at 100,000×g at 4° C. and aliquots of the supernatant werestored at −80° C. before analysis.

In the following human IgG drug level sandwich ELISA the humanNI-302.35C1, NI-302.11F11 antibody plasma levels were determined usingthe corresponding recombinant antibody of known concentration asstandard. 96 well microplates (Corning) were coated with donkeyantihuman IgG (709-005-149, Jackson Immunoresearch) at 1 μg/ml in 50 mMcarbonate coating buffer pH 9.6. Non-specific binding sites were blockedfor 1 hr at RT with PBS/0.1% Tween®-20 containing 2% BSA (Sigma, Buchs,Switzerland). Plasma samples were diluted 1:20,000 and 1:100,000, brainhomogenates were diluted 1:5 and 1:50 and both were incubated 1 hr at RTtogether with the standard dilution curves. Binding was determined usingthe detection antibody anti human HRP (709-036-098, JacksonImmunoresearch), followed by measurement of HRP activity in a standardcolorimetric assay. Concentrations of plasma and spinal cord sampleswere calculated based on the individual standard curve. Values shown intable 6-1 are average values of 2 independent ELISA experiments.

Plasma and brain samples were obtained 2 days after a singleintraperitoneal injection of 50 mg/kg of antibodies NT-302.31F11,NT-302.35C1 in R6/1 transgenic mice. Antibody levels in plasma and brainhomogenates were determined by human sandwich IgG ELISA (FIGS. 18 A andB). The ratio of brain versus plasma drug levels was determined at0.13±0.02% and 0.21±0.06% for human-derived antibodies NI-302.31F11 andNI-302.35C1, respectively. These results suggest a 48h brain penetrationof the tested NI-302 antibodies in the expected range in HTT transgenicmice.

Example 25: Characterization of Binding Affinity and Specificity ofFurther Antibodies Identified in Accordance with the Present Invention

To determine the half maximal effective concentration (EC₅₀) of furtheridentified recombinant human-derived HTT antibodies to soluble andaggregated HTT Exon1 proteins with 21 or 49 polyQ repeats direct ELISAwith coating of the different preparations at 5 μg/ml concentration andEC₅₀ determination was performed as described in Example 6, supra.

The determined EC₅₀ for the different HTT species are shown in FIG. 19as well as FIG. 31 (A, D-F) and summarized in FIG. 20. Mosthuman-derived antibodies bound with high affinity at subnanomolar or lownanomolar EC₅₀. Some candidates such as NI-302.37C12, NI-302.55D8,NI-302.11A4, NI-302.22H9 or NI-302.64E5 seemed to have preferred bindingto uncut GST-HTT protein, other antibodies such as NI-302.74C11,NI-302.71F6, NI-302.4A6, NI-302.12H2 or NI-302-8M1 showed high affinitybinding with similar EC₅₀-values to all HTT preparation in the ELISAassay. NI-302.15F9 showed about a 5-fold preference of HD49 vs. HD21with EC₅₀ values in the range of 5 to 35 nM. Antibody 33C11 served ascontrol in this experiment (FIG. 32 G).

Therefore, a panel of high affinity recombinant HTT specific humanantibodies was cloned from memory B-cells derived from healthy elderlyhuman donor cohorts and recombinantly expressed and characterized.Additionally, a screening campaign for additional backup antibodies wasinitiated in a cohort of selected presymptomatic patients withHuntington's disease (HD) carrying different lengths CAG repeatexpansions.

To further characterize the binding of the identified recombinanthuman-derived HTT NI-302 antibodies to soluble and aggregated HTT Exon1proteins with 21, 35 or 49 polyQ repeats filter retardation assayutilizing 0.2 μg/ml primary antibody and dot-blot analysis utilizing 1μg/ml primary antibody were performed as described in Example 7, supra.

As shown in FIG. 21 and FIG. 32 (A, G), on the dot blot (Fig. left side,DotBlot) most of the antibodies characterized showed a preference fordetection of HTT proteins with expanded polyQ tracts (HD49>>HD35>HD21).Furthermore, the signal intensity increased with increasing incubationtimes of the aggregation reactions of HD35 and HD49 in particular forantibodies NI-302.15F9, NI-302.71F6 (FIG. 21, first row of blots) andNI-302-64E5 (FIG. 32 A, left side). In the filter retardation analysisNI-302.15F9, NI-302.71F6 (FIG. 21, right side, FRA) and NI-302-64E5(FIG. 32 A, right side) detected SDS stable HD35 and HD49 aggregatesthat were retained on the 0.2 μm pore size membrane whereas otherantibodies such as NI-302.44D7 and NI-302.37C12 (FIG. 21, second row ofblots) or NI-302.4A6, NI-302.12H2 and NI-302.8M1 (FIG. 32 D, E, F) didnot bind to aggregates on the filter. Antibody 33C11 served as controlin this experiment (FIG. 32 G). These findings based on spotted proteinpreparations suggest that several of the cloned NI-302 antibodies show apreference for aggregated HTT conformations with pathogenic polyQexpansions.

Additionally, the binding specificity of the identified antibodies tounrelated proteins, in particular to proteins forming aggregates wasassessed by direct ELISA (FIG. 22 and FIG. 33 A, D-F) as alreadydescribed in Example 8, supra. The results showed that most of thehuman-derived NI-302 antibodies tested bind specifically to HTT withabsent binding to the other unrelated proteins tested.

Example 26: Assessment of the Binding and Epitope Mapping ofHuman-Derived HTT Antibodies

To map the HTT epitope recognized by the newly identified human-derivedantibodies epitope mapping with synthetic peptides was performed asdescribed above in Example 9 (FIG. 23 and FIG. 35 A, D-F). Additionally,the half maximal effective concentration (EC₅₀) of the HTT antibodies toBSA-coupled peptide fragments of the HTT Exon 1 by direct ELISA withBSA-coupled Htt Exon1 domain peptides was determined as well as EC₅₀determination were performed as described in Example 10.

Example 27: Assessment of the Binding of HTT Antibodies in Human HTTTransgenic Mice

The characterization of the binding of the identified antibodies to HTTpathology in human HTT transgenic mouse brain tissues was assessed asdescribed in Example 12, supra with the difference that the incubationof the sections was performed with 5 nM ffig.3M (74C11, 39C12, 11A4,22H9, 78H12, 37C12, 7D8, 72F10) or 50 nM concentrations (15F9, 71F6,55D8, 44D7, 7A8, 64E5) of the anti-HTT antibodies. As shown in FIG. 24the identified human-derived anti-HTT antibodies revealed a prominentand highly specific staining of neuronal intranuclear inclusionpathology in the striatum and cortex of R6/1 transgenic animals, as alsoshown for the antibodies NI-302.33C11, NI-302.63F3, and NI-302.35C1,described above. These findings are consistent with the high affinitybinding to HTT aggregates as determined by ELISA and dot blot analysisin Example 26.

Example 28: Basic Characterization of Transgenic Mouse Model R6/1 ofHuntington's Disease (HD)

Tg(HDexon1)61Gpb/J transgenic mice harbor a 1.9 kb transgene which wasisolated from a phage genomic clone derived from an HD patient andcontained the 5′ end of the human huntingtin (HTT) gene. It was composedof approximately 1 kb of 5′ UTR sequences, exon 1 (carrying expanded CAGrepeats of ˜130 units) and the first 262 bp of intron 1. This constructwas microinjected into single cell CBAxC57BL/6 embryos. Male founder R6was bred to CBAxC57BL/6 females, producing several founder lines(Mangiarini et al., Cell 87 (1996), 493-506). Mice from founder lineR6/1 have the transgene integrated as a single intact copy which isubiquitously expressed. Transgenic mice on a mixed CBAxC57BL/6 geneticbackground were backcrossed to C57BL/6J for more than 12 to generate thecongenic strain B6.Cg-Tg(HDexon1)61Gpb/J. R6/1 transgenic mice exhibit aprogressive neurological phenotype that mimics many of the features ofHD, including choreiform-like movements, involuntary stereotypicmovements, tremor, and epileptic seizures, as well as nonmovementdisorder components, including unusual vocalization. They urinatefrequently and exhibit loss of body weight and muscle bulk through thecourse of the disease. Neurologically they develop neuronal intranuclearinclusions (NII) which contain both the HTT and ubiquitin proteins.These NII have also been identified in human HD patients. The age ofonset of HD symptoms is reported to occur between 15 and 21 weeks forthis 6/1 line (Naver et al, Neuroscience 122 (2003), 1049-1057; Hodgeset al, Genes Brain Behav. 7(3) (2008), 288-299).

R6/1 transgenic mice obtained from Jackson Laboratories were expandedand longitudinally characterized with respect to behavior phenotype,longitudinal development of body weight, total brain weight,histopathological analysis and survival, as shown in FIG. 25. Thefindings obtained by in large were in line with the published data andidentified this mouse line as a suitable preclinical model for efficacystudies with human-derived NI-302 antibodies targeting aggregated HTT.

Example 29: Basic Characterization of Transgenic Mouse Model N171-82Q ofHD

The B6C3-Tg(HD82Gln)81Dbo/J (N171-82Q) transgenic mouse line (Schillinget al., Hum Mol Genet. 8(3) (1999), 397-407) is a well characterizedmouse model for HD. B6C3-Tg(HD82Gln)81Dbo/J (N171-82Q) transgenic miceexpresses an N-terminally truncated human HTT cDNA that encodes 82glutamines and encompasses the first 171 amino acids. The altered HTTcDNA is under control of a mouse prion protein promoter. Expression isobserved in neurons of the central nervous system. Mice expressing thistransgene appear normal at birth through 1-2 months. However, the micefail to gain weight, develop tremors, hypokinesis and lack coordination.They exhibit an abnormal gait and frequent hind limb clasping. Theirlife expectancy is 5-6 months. Studies using HTT antibodies indicateddiffuse nuclear labeling and numerous immunoreactive nuclear inclusionsin multiple neuron populations. Additionally neuritic damage wasevident.

N171-82Q transgenic mice obtained from Jackson Laboratories wereexpanded and longitudinally characterized with respect to behaviorphenotype, longitudinal development of body weight, total brain weightat endstage, histopathological analysis and survival (FIG. 26). Thesefindings by in large were in line with published data and identifiedthis mouse line additionally to the mouse line described in Example 29,as a suitable preclinical model for efficacy studies with human-derivedNI-302 antibodies targeting aggregated HTT.

Example 30: Assessment of Neuronal Inclusion Staining in Huntington'sDisease (HD) Patients

To assess the staining of neuronal inclusions by the identifiedantibodies of the present invention in patients immunochistochemicalanalysis was performed. The assessment of the binding of identifiedantibodies to HTT pathology in human brain tissues was assessed asdescribed in Example 12, supra with the difference that the incubationof the sections was performed with 50 nM of NI-302.33C11, 50 nM ofNI-302.63F3 or 100 nM of NI-302.35C1 antibody.

As shown in FIG. 27 the immunohistochemical analysis with thepolyP-region binding antibody NT-302.33C11 showed a staining of neuronalintranuclear inclusions in cortical neurons of Huntington Diseasepatients (FIG. 27 A-D) at 50 nM and in striatal neurons of 270 day old,late disease stage B6.Cg-Tg(HDexon1)61Gpb/J) transgenic animals at 1 (E)and 5 nM (F) concentration, while no staining was detected innon-transgenic littermates (G), when the primary antibody was omittedduring the staining (H) or if tissue of non-Huntington Disease controlswas stained with 50 nM of NI-302.33C11. The P-rich-domain antibodyNI-302.63F3 (FIG. 28) and anti-C-terminal domain antibody NI-302.35C1(FIG. 29) showed within the immunohistochemical analysis with 50 nM ofNI-302.63F3 or 100 nM of NI-302.35C1 a staining of neuronal intranuclearinclusions (A-C) and staining of some neurites (D) of cortical neuronsof four different Huntington Disease patients (A-D). A staining couldalso be observed in the striatal neurons of 270 day old, late diseasestage B6.Cg-Tg(HDexon1)61Gpb/J) transgenic animals at 1 (E) and 50 nM(F) concentration. No staining was detected in non-transgeniclittermates (G), if primary antibody was omitted during the staining (H)or if tissue of non-Huntington Disease controls was stained with 50 nMof NI-302.63F3 or 100 nM of NI-302.35C1, respectively.

In contrast to the specific anti-HTT antibodies of the presentinvention, immunohistochemical analysis with the commercially availableanti-polyQ antibody Mab1574 (1:2000, Chemicon) showed additionalstaining of tissue, i.e. a more general nuclear and cytoplasmic stainingand staining of some neurites (FIG. 30 A, D) of cortical neurons of fourdifferent Huntington Disease patients and in striatal neurons ofpresymptomatic, 150 day old (FIG. 30 E) and 270 day old (FIG. 30 F),late disease stage B6.Cg-Tg(HDexon1)61Gpb/J) transgenic animals.

Example 31: Characterization of Binding Affinity and Selectivity ofAnti-Poly Q/P Domain NI-302.7D8 Antibody Utilizing Direct ELISA and EC₅₀

To determine the half maximal effective concentration (EC₅₀) ofrecombinant human-derived HTT antibody NI-302.7D8 to soluble andaggregated HTT Exon1 proteins with 21 or 49 polyQ repeats direct ELISAand EC50 determination was performed as described in Example 6, supra.

It could be shown that NI-302.7D8 binds with similar high affinity tosoluble GST-HD21 and aggregated HD21 with an EC₅₀ of approximately 50 to100 nM albeit showing a preference to the elongated more pathogenic formof aggregated HD49 and soluble GST-HD49 with an EC₅₀ of 17 and 6 nMrespectively (FIG. 31 B)

Accordingly, the human-derived HTT anti-poly Q/P domain antibodyNI-302.7D8 targets an epitope exposed in aggregated as well as in anuncut, more linear structure of HTT Exon1 protein with high-affinity inthe low nanomolar range.

Additionally, to characterize the binding of recombinant human-derivedHTT antibody NI-302.7D8 to soluble and aggregated HTT Exon1 proteinswith 21, 35 or 49 polyQ repeats using filter retardation assay and dotblot at a concentration of 1 ug/ml. as described in Example 7, supra.

It could be shown that on the dot blot (FIG. 32 B, left side), antibodyNI-302.7D8 equally good detected proteins of huntingtin with expandedpolyQ tracts (HD49=HD35=HD21). In the filter retardation analysis (FIG.32 B, right side, FRA) NI-302.7D8 did not bind to SDS stable HD21, HD35or HD49 aggregates on the filter membrane.

Furthermore, to determine the binding of NI-302.7D8 recombinant antibodyto unrelated aggregating protein targets, direct ELISA was performed asdescribed in Example 8, supra. As shown in FIG. 33 B human-derivedNI-302.7D8 bound specifically to HTT while a binding to unrelatedproteins could not be shown.

Example 32: Assessment of the Binding Epitope of the HTT AntibodiesNI-302-64E5, NI-302.7D8 and NI-302.72F10

To map the huntingtin epitope recognized by the NI-302-64E5, NI-302.7D8and NI-302.72F10 human-derived antibody epitope mapping with syntheticpeptides was performed as described above in Example 9.

FIG. 35 A shows a prominent binding of NI-302-64E5 to peptides number 10to 12 indicating that the epitope recognized by this antibody islocalized in the P-rich repeat domain of huntingtin. The NI-302.64E5binding epitope is therefore predicted to be localized within HTT aminoacids 48-PQPPPQAQPL-58 (SEQ ID No.: 200). As shown in FIG. 35 B,prominent binding of NI-302.7D8 was observed to peptides number 6 to 8indicating that the epitope recognized by this antibody is localized inthe polyQ/polyP repeat domain of huntingtin. The NI-302.7D8 bindingepitope is therefore predicted to be localized within HTT amino acids28-QQQQQQQPPP-37 (SEQ ID No.: 201). In contrast, prominent binding ofNI-302.72F10 was observed to peptides number 15 and 16 indicating thatthe epitope recognized by this antibody is localized at theanti-N-terminal domain of HTT (FIG. 35 C). The NI-302.72F10 bindingepitope was therefore predicted to be localized within HTT amino acids70-PPPGPAVAEEPLH-82 (SEQ ID No.: 202).

Example 32: Characterization of Binding Affinity and Selectivity ofAnti-N-Terminal Domain Antibody NI-302.15E8 Utilizing Direct ELISA andEC₅₀

To determine the half maximal effective concentration (EC₅₀) ofrecombinant human-derived HTT antibody NI-302.15E8 to soluble andaggregated HTT Exon1 proteins with 21 or 49 polyQ repeats direct ELISAand EC₅₀ determination was performed as described in Example 6, supra.

It could be shown that NI-302.15E8 binds with higher affinity tonon-aggregated GST-HD49 and GST-HD21 and less affinity to aggregatedHD49 and HD21, see FIG. 14 A and B. Accordingly, the human-derived HTTanti-N-terminal domain antibody NI-302.15E8 target an epitope exposed inboth aggregated as well as in soluble forms of HTT, albeit with a higheraffinity to soluble forms of HTT.

Example 33: Epitope Mapping by Direct ELISA Binding to Different Exon1Peptides of the HTT Antibody NI-302.15E8

To determine the half maximal effective concentration (EC50) ofrecombinant human-derived HTT antibody NI-302.15E8 to BSA-coupledpeptide fragments of the huntingtin Exon 1 direct ELISA with BSA-coupledHtt Exon1 domain peptides and EC₅₀ determination were performed asdescribed in Example 10.

As shown in FIG. 15 NI-302.15E8 binds with high affinity to the first 19BSA-coupled amino acids at the N-terminus as well as to full-lengthGST-HD49 with an EC₅₀ of approx. 0.1 or 15, respectively.

Example 34: Impact of HTT Antibody NI-302.35C1 on Behavioral Deficits inHuman HTT Transgenic Mice

An Elevated Plus Maze test to measure anxiety-like behavior and a Poletest to measure motor performance and coordination in human HTT micewere performed to study the anti-HTT antibody NI-302.35C1 in vivo.

Groups and Treatment

For the behavioral analysis two groups of mice with n=24 (12/12male/female) B6.Cg-Tg(HDexon1)61Gpb/J transgenic (tg) mice as describedin Example 12 and one group of wild type (wt) mice were used. The groupsof transgenic mice received intraperitoneal treatment of either 30 mg/kgmouse chimeric NI-302.35C1 or vehicle starting at an age for 6-7 weeksuntil end stage phenotype of the mice between 7 to 9 months of age andthe wild type mice were injected with the same volume of vehicle. TheElevated-Plus-Maze and pole test behavioral tests were performed at anage of 16 and 18 weeks of age respectively.

Elevated Plus Maze Test

The Elevated Plus Maze test was performed according to Naver et al.,Neursoscience 122 (2003), 1049-1057. The maze was elevated 50 cm abovethe floor. Four maze arms (30 cm×5 cm) originated from a centralplatform forming a cross. Two of the arms located opposite each otherwere enclosed by 15 cm high walls (closed arms) while the other arms didnot have any kind of screening (open arms). The test were performed atthe beginning of the dark phase of the animals and the illumination onthe open arm was in the range of 40 lux. Each mouse was placed in thecenter of the Plus Maze facing an open arm. The experiment lasted for 5min and was recorded with a videotracking system (VideoMot Software, TSESystems). Between the sessions, the maze was rinsed with water and driedwith a paper towel. Subsequently the number of entries made into openand closed arms as well as the time spent in open and closedcompartments were evaluated. An entry was defined as all four paws inone arm. The number of entries into the open arms and the time spent inthe open arms are used as indices of open space-induced anxiety in mice.

Pole Test

The pole test is used widely to assess basal ganglia-related movementdisorders in mice; see, e.g. Matsuura et al. J. Neurosci. 73 (1997),45-48; Sedelis et al. Behav Brain Res. 125 (2001), 109-125, Fernagut etal., Neuroscience 116 (2003), 1123-1130. Briefly, animals were placedhead-up on top of a vertical wooden pole 50 cm long (1 cm in diameter).The base of the pole was placed in the home cage. When placed on thepole, animals orient themselves downward and descend the length of thepole back into their home cage. On the test day, animals received threetrials, and the total time to descend (t-total) were measured. Theresults of the third trial of the day is shown in FIG. 24 B.

As shown in FIG. 34 A NI-302.35C1 treated R6/1 animals spend less timein the open arms, entered the open arms less frequently and did lessunprotected head dips on the open arm compared to vehicle treated R6/1animals. Hence the NI-302.35C1 treated R6/1 mice displayed a moreanxious phenotype, comparable to the non-transgenic littermates.Furthermore, as shown in FIG. 24 B NI-302.35C1 treated R6/1 animalshowed an improved performance in the pole test compared to vehicletreated R6/1 animals reaching levels similar to non-transgenic animals.In summary, the antibody NI-302.35C1 of the present invention has abeneficial impact on behavioral performance and motor-related tasks inhuman HTT transgenic mice.

Example 35: Sequence Alignment of HTT Antibodies

The determination of percent identity or similarity was performed withthe standard parameters of the BLASTn program as described in section“Definitions” of the present invention. As shown in FIG. 36 allantibodies of the present invention are rich of tyrosines in the CDRs.

Example 36: Generation of Bispecific Anti-HTT Antibodies

The generation of bispecific antibodies can be performed as generallydescribed in Brennan; see supra. Starting material for producingbispecific antibodies are intact IgG anti-HTT antibodies of the presentinvention recognizing either a polyP-region, a polyQ/polyP-region, theP-rich-region, the C terminal-region or the N-terminal region of HTTexon 1 protein as described in the Examples and summarized in FIG. 20.The antibodies are treated with pepsin for three hours at 37° C. treatedin acetate buffer pH 4.0, to cleave the Fc portion of the antibody. Thereaction is stopped by increasing the pH to 8 with Tris buffer.Subsequently the solution is filled up with an equal volume of a mixtureof 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB) and incubated withthionitrobenzoate (TNB) for 20 hours at room temperature. The molarratio of the DTNB-TNB mixture is 20:30 being established by incubating a40 mM DTNB solution with a 10 mM DTT solution for several minutes. Afterfurther reduction of the two modified F(ab′) fragments with 0.1 mM DTTfor one hour at 25° C., the thus obtained F(ab′)-TNB and F(ab′)-SHfragments are hybridized to a bispecific F(ab′)₂-fragment for 1 h at 25°C. Bispecific F(ab′)₂-fragments were purified via gel filtration(Superdex 200 column).

Example 37: Characterization of Binding Affinity and Selectivity ofBispecific Anti-HTT Antibodies Utilizing Direct ELISA and EC50

To determine the half maximal effective concentration (EC₅₀) ofbispecific HTT antibodies to soluble and aggregated HTT Exon1 proteinswith 21 or 49 polyQ repeats direct ELISA and EC₅₀ determination isperformed as described in Example 6, supra. The bispecific HTTantibodies bind with high affinity to all four species including theaggregated HTT Exon1 HD49, equally targeted their respective epitopesexposed in aggregated as well as in soluble forms of HTT with lownanomolar affinity. Additionally, to characterize the binding ofbispecific HTT antibodies to soluble and aggregated HTT Exon1 proteinswith 21, 35 or 49 polyQ repeats filter retardation assay and dot blot asdescribed in Example 7, supra, are performed. On the dot blot,bispecific HTT antibodies preferentially detect constructs of HTT withexpanded polyQ tracts. Furthermore, the signal intensity increases withincreasing incubation times of the aggregation reactions of HD35 andHD49. In the filter retardation analysis bispecific HTT antibodiesdetect HD35 and HD49 aggregates that are retained on the 0.2 μm poresize membrane. Based on their dual specificity to HTT and the previousfindings for the binding of the individual antibodies on membrane boundprotein preparations it is expected that bispecific HTT antibodiespreferentially target aggregated HTT conformations with pathogenic polyQexpansions. Furthermore, to determine the binding of bispecific anti-HTTantibodies to unrelated aggregating protein targets, direct ELISA isperformed as described in Example 8, supra. In this context, bispecificanti-HTT antibodies bind specifically to HTT while a binding tounrelated proteins may not be shown.

1. A human-derived monoclonal anti-huntingtin (HTT) antibody or anHTT-binding fragment, synthetic or biotechnological derivative thereof,which comprises variable domains of both the light (V_(L)) and heavy(V_(H)) chain and a constant domain.
 2. The antibody of claim 1, whichis of the IgG type.
 3. The antibody of claim 1 or 2, wherein the lightchain is kappa (κ).
 4. The antibody of any one of claims 1 to 3, whichrecognizes an epitope in the amino acid sequence of exon 1 of the HTTgene, preferably an epitope in a polyP-region, the P-rich-region, theC-terminal region, the N-terminal-region, the Q/P-rich-region or aconformational epitope of HTT exon
 1. 5. The antibody of any one ofclaims 1 to 4, which is capable of binding a peptide comprising theepitope and/or aggregated forms of HTT exon
 1. 6. The antibody of anyone of claims 1 to 5 or an HTT-binding fragment, synthetic orbiotechnological derivative thereof which specifically binds (i) anepitope in a polyP-region which comprises the amino acid sequencePPPPPPPPPPP (SEQ ID Nos.: 146, 147, 148, 149, 150, 152, 153, 155, 156),PPPPPPPP (SEQ ID Nos. 139, 151, 154, 158, 161), PPPPPP (SEQ ID Nos.:157, 159, 160); (ii) an epitope in the P-rich-region which comprises theamino acid sequence PQPPPQAQPL (SEQ ID No. 140), PPPQLPQPPP (SEQ ID No.141), QAQPLLPQPQPPPPP (SEQ ID No. 142), or PPPQLPQPPPQAQPL (SEQ ID No.143); (iii) an epitope in the C-terminal region of exon 1 of HTT whichcomprises the amino acid sequence PPGPAVAEEPLHRP (SEQ ID No.: 145); orPPGPAVAEEPLH (SEQ ID No. 202) (iv) an epitope in the N-terminal regionof exon 1 of HTT which comprises the amino acid sequence KAFESLKSFQQ(SEQ ID NO: 144); (v) an epitope in the Q/P-rich-region which comprisesthe amino acid sequence QQQQQQQQQPPP (SEQ ID No. 201); or (vi) aconformational epitope which is not present on a linear peptide derivedfrom HTT exon 1 but on aggregated recombinant HTT exon1 proteinsHttExon1Q21 (HD21) and HttExon1Q49 (HD49).
 7. The antibody of claim 6 oran HTT-binding fragment, synthetic or biotechnological derivativethereof, which is selected from the group consisting of (i) an antibodyrecognizing a polyP-region of HTT comprising in its variable region (a)at least one complementarity determining region (CDR) of the V_(H)and/or V_(L) variable region amino acid sequence of any one ofantibodies NI-302.33C11, NI_302.74C11, NI-302.15F9, NI-302.39G12,NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8,NI-302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8,NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9 shown in FIG. 1A, 1G,1H, 1I, 1J, 1H, 1L, 1M, 1N, 10, 1P, 1Q, 1R, 1S, 1T, 1U, 1V, 1W, 1AD,1AJ, 1AK, 1AL, 1AM, 1AN or 1AO and depicted in (i) V_(H) sequence (SEQID NOs: 1, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81,85, 89); and (ii) V_(L) sequence (SEQ ID NOs: 3, 27, 31, 35, 39, 43, 47,51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91), respectively; (b) an aminoacid sequence of the V_(H) and/or V_(L) region of any one of antibodiesNI-302.33C11, NI_302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4,NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8,NI-302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1,NI-302.8F1, NI-302.52C9, NI-302.46C9 as depicted in FIG. 1A, 1G, 1H, 1I,1J, 1H, 1L, 1M, 1N, 10, 1P, 1Q, 1R, 1S, 1T, 1U, 1V, 1W, 1AD, 1AJ, 1AK,1AL, 1AM, 1AN or 1AO; (c) at least one CDR consisting of an amino acidsequence resulted from a partial alteration of any one of the amino acidsequences of (a); (d) a heavy chain and/or a light variable regioncomprising an amino acid sequence resulted from a partial alteration ofthe amino acid sequence of (b); and/or (e) the antibody or antigenbinding fragment thereof optionally further comprising a polypeptidesequence which is heterologous to the V_(H) and/or V_(L) region or theleast one CDR, preferably wherein polypeptide sequence comprises a humanconstant domain, preferably of the IgG type, most preferably of the IgG1class or isotype; (ii) an antibody recognizing the P-rich-region of HTTcomprising in its variable region (a) at least one complementaritydetermining region (CDR) of the V_(H) and/or V_(L) variable region aminoacid sequence of any one of antibodies NI-302.63F3, NI-302.31F11,NI-302.2A2, NI-302.15D3 or NI-302.64E5 shown in FIG. 1B, 1D, 1E, 1X,1AE, 1AG, 1AH, 1AQ, 1AR or 1AS and depicted in (i) V_(H) sequence (SEQID NOs: 5, 13, 17, 135, 164, 166); and (ii) V_(L) sequence (SEQ ID NOs:7, 15, 19, 137, 168), respectively; (b) an amino acid sequence of theV_(H) and/or V_(L) region of any one of antibodies NI-302.63F3,NI-302.31F31, NI-302.2A2, NI-302.15D3 or NI-302.64E5 as depicted in FIG.1B, 1D, 1E, 1X, 1AE, 1AG, 1AH, 1AQ, 1AR or 1AS; (c) at least one CDRconsisting of an amino acid sequence resulted from a partial alterationof any one of the amino acid sequences of (a); (d) a heavy chain and/ora light variable region comprising an amino acid sequence resulted froma partial alteration of the amino acid sequence of (b); and/or (e) theantibody or antigen binding fragment thereof optionally furthercomprising a polypeptide sequence which is heterologous to the V_(H)and/or V_(L) region or the least one CDR, preferably wherein polypeptidesequence comprises a human constant domain, preferably of the IgG type,most preferably of the IgG1 class or isotype; (iii) an antibodyrecognizing the C-terminal-region of HTT exon 1 comprising in itsvariable region (a) at least one complementarity determining region(CDR) of the V_(H) and/or V_(L) variable region amino acid sequence ofantibody NI-302.35C1 or NI-302.72F10 shown in FIG. 1C, 1Z, 1AF or 1ATand depicted in (i) V_(H) sequence (SEQ ID NO: 9, 176 or 178); and (ii)V_(L) sequence (SEQ ID NO: 11, 180 or 182), respectively; (b) an aminoacid sequence of the V_(H) and/or V_(L) region of antibody NI-302.35C1or NI-302.72F10 as depicted in FIG. 1C, 1Z, 1AF or 1AT; (c) at least oneCDR consisting of an amino acid sequence resulted from a partialalteration of any one of the amino acid sequences of (a); (d) a heavychain and/or a light variable region comprising an amino acid sequenceresulted from a partial alteration of the amino acid sequence of (b);and/or (e) the antibody or antigen binding fragment thereof optionallyfurther comprising a polypeptide sequence which is heterologous to theV_(H) and/or V_(L) region or the least one CDR, preferably whereinpolypeptide sequence comprises a human constant domain, preferably ofthe IgG type, most preferably of the IgG1 class or isotype; (iv) anantibody recognizing the N-terminal-region of HTT comprising in itsvariable region (a) at least one complementarity determining region(CDR) of the V_(H) and/or V_(L) variable region amino acid sequence ofantibody NI-302.15E8 shown in FIG. 1AP and depicted in (i) V_(H)sequence (SEQ ID NOs: 131); and (ii) V_(L) sequence (SEQ ID NOs: 133),respectively; (b) an amino acid sequence of the V_(H) and/or V_(L)region of antibody NI-302.15E8 as depicted in FIG. 1AP; (c) at least oneCDR consisting of an amino acid sequence resulted from a partialalteration of any one of the amino acid sequences of (a); (d) a heavychain and/or a light variable region comprising an amino acid sequenceresulted from a partial alteration of the amino acid sequence of (b);and/or (e) the antibody or antigen binding fragment thereof optionallyfurther comprising a polypeptide sequence which is heterologous to theV_(H) and/or V_(L) region or the least one CDR, preferably whereinpolypeptide sequence comprises a human constant domain, preferably ofthe IgG type, most preferably of the IgG1 class or isotype; (v) anantibody recognizing the Q/P-rich-region of HTT comprising in itsvariable region (a) at least one complementarity determining region(CDR) of the V_(H) and/or V_(L) variable region amino acid sequence ofantibody NI-302.7D8 in FIG. 1Y and depicted in (i) V_(H) sequence (SEQID NOs: 172); and (ii) V_(L) sequence (SEQ ID NOs: 174), respectively;(b) an amino acid sequence of the V_(H) and/or V_(L) region of antibodyNI-302.7D8 as depicted in FIG. 1Y; (c) at least one CDR consisting of anamino acid sequence resulted from a partial alteration of any one of theamino acid sequences of (a); (d) a heavy chain and/or a light variableregion comprising an amino acid sequence resulted from a partialalteration of the amino acid sequence of (b); and/or (e) the antibody orantigen binding fragment thereof optionally further comprising apolypeptide sequence which is heterologous to the V_(H) and/or V_(L)region or the least one CDR, preferably wherein polypeptide sequencecomprises a human constant domain, preferably of the IgG type, mostpreferably of the IgG1 class or isotype; and (vi) an antibodyrecognizing a conformational epitope which is not present on a linearpeptide derived from HTT exon 1 but on aggregated recombinant HTT exon1proteins HttExon1Q21 (HD21) and HttExon1Q49 (HD49) comprising in itsvariable region (a) at least one complementarity determining region(CDR) of the V_(H) and/or V_(L) variable region amino acid sequence ofany one of antibodies NI-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1shown in FIG. 1F, 1AA, 1AB, 1AC or 1AU and depicted in (i) V_(H)sequence (SEQ ID NOs: 21, 184, 188, 190, 194 or 196); and (ii) V_(L)sequence (SEQ ID NOs: 23, 186, 192 or 198), respectively; (b) an aminoacid sequence of the V_(H) and/or V_(L) region of any one of antibodiesNI-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1 as depicted in FIG.1F, 1AA, 1AB, 1AC or 1AU; (c) at least one CDR consisting of an aminoacid sequence resulted from a partial alteration of any one of the aminoacid sequences of (a); (d) a heavy chain and/or a light variable regioncomprising an amino acid sequence resulted from a partial alteration ofthe amino acid sequence of (b); and/or (e) the antibody or antigenbinding fragment thereof optionally further comprising a polypeptidesequence which is heterologous to the V_(H) and/or V_(L) region or theleast one CDR, preferably wherein polypeptide sequence comprises a humanconstant domain, preferably of the IgG type, most preferably of the IgG1class or isotype.
 8. The antibody of any one of claims 1 to 7, whereinthe antibody has a binding affinity corresponding to an EC50 (halfmaximal effective concentration) value of ≤20 nM, preferably ≤10 nM andmost preferably ≤1 nM for binding HD49 and an EC50 value of 40 nM,preferably ≤10 nM and most preferably ≤1 nM for binding HD21.
 9. Theantibody of any one of claims 1 to 8 which is a chimeric murine-human ora murinized antibody and/or an antibody or HTT-binding molecule whichcompetes with an antibody of any one of claims 1 to 4 for specificbinding to HTT.
 10. A polynucleotide encoding at least the bindingdomain or variable region of an immunoglobulin chain of the antibody ofany one of claims 1 to 9, preferably wherein the polynucleotide is acDNA.
 11. A vector comprising the polynucleotide of claim 10, optionallyin combination with a polynucleotide of claim 6 that encodes thevariable region of the other immunoglobulin chain of said bindingmolecule.
 12. A host cell comprising a polynucleotide of claim 10 or avector of claim
 11. 13. A method for preparing an anti-HTT antibody, abiotechnological derivative, or immunoglobulin chain(s) thereof, saidmethod comprising (a) culturing the cell of claim 12 and (b) isolatingthe antibody or immunoglobulin chain(s) thereof from the culture.
 14. Anantibody or immunoglobulin chain(s) thereof encoded by a polynucleotideof claim 10, a vector of claim 1 or obtainable by the method of claim13.
 15. The antibody of any one of claims 1 to 9 or 14, which is abispecific antibody.
 16. The antibody of claim 15, which recognizes twodifferent epitopes on a protein encoded by exon 1 of the HTT gene,preferably wherein at least one epitope is selected form the groupconsisting of the epitopes as defined in any one of claims 4 to
 7. 17.The antibody of any one of claims 1 to 9 or 14 to 16, which is (i)detectably labeled, preferably wherein the detectable label is selectedfrom the group consisting of an enzyme, a radioisotope, a fluorophore,and a heavy metal; or (ii) attached to a drug.
 18. A compositioncomprising the antibody of any one of claims 1 to 9 or 14 to 17, thepolynucleotide of claim 10, the vector of claim 11, the cell of claim12, preferably wherein the composition is (a) a pharmaceuticalcomposition and further comprises a pharmaceutically acceptable carrier,optionally further comprising an additional agent useful for treatingdiseases and/or symptoms associated with HTT amyloidosis, preferablywherein the composition is a vaccine; (b) a diagnostic composition,preferably which comprises reagents conventionally used in immuno ornucleic acid based diagnostic methods.
 19. A HTT binding moleculecomprising at least one CDR of an antibody of any one of claims 1 to 9or 14 to 17 for use in in vivo detection of or targeting a therapeuticand/or diagnostic agent to HTT in the human or animal body, preferablywherein said in vivo imaging comprises positron emission tomography(PET), single photon emission tomography (SPECT), near infrared (NIR),optical imaging or magnetic resonance imaging (MRI).
 20. A peptidehaving an epitope of HTT specifically recognized by an antibody of anyone of claims 1 to 9 or 14 to 17, preferably wherein the peptidecomprises an amino acid sequence as defined in claim 2 or a modifiedsequence thereof in which one or more amino acids are substituted,deleted and/or added, wherein the peptide is recognized by the antibodyof any one of claims 1 to 9 or 14 to
 17. 21. A kit useful in thediagnosis or monitoring of disorders associated with HTT amyloidosis,said kit comprising at least one antibody of any one of claims 1 to 9 or14 to 17 or a HTT binding molecule having substantially the same bindingspecificities of any one thereof, the polynucleotide of claim 10, thevector of claim 11 or the cell of claim 12 and/or the peptide of claim220, optionally with reagents and/or instructions for use.
 22. A methodof treating a disease and/or symptoms associated with HTT amyloidosis orfor in in vivo detection of or targeting a therapeutic and/or diagnosticagent to HTT in the human or animal body comprising administering to asubject in need thereof a therapeutically or diagnostically effectiveamount of an anti-HTT antibody or an HTT-binding fragment, synthetic orbiotechnological derivative thereof, which comprises a variable light(V_(L)) and heavy (V_(H)) chain, and a constant domain for use intreating a disease and/or symptoms associated with HTT amyloidosis orfor in in vivo detection of or targeting a therapeutic and/or diagnosticagent to HTT in the human or animal body.
 23. The method of claim 22,wherein the anti-HTT antibody is an antibody of any one claims 1 to 9 or14 to 17.